CN108277167A - A kind of method of biological control Phytophthora capsici disease - Google Patents
A kind of method of biological control Phytophthora capsici disease Download PDFInfo
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Abstract
The invention belongs to biotechnologies, disclose a kind of method of biological control Phytophthora capsici disease comprising following steps：Biological agent is added in the water of 100 200 times of weight, then addition accounts for the glucose of 23 times of weight of bacteria agent and accounts for the urea of 12 times of weight of biological agent, 200rpm stirs 3min, it is subsequently placed in 28 30 DEG C of 3 5h of activation, pepper seedling is immersed into 30 60min of aforesaid liquid again, pepper seedling is taken out, soil is implanted into.The method of the present invention is environment friendly and pollution-free, and Phytophthora capsici can be effectively prevent sick.
Description
Technical field
The invention belongs to biotechnologies, and in particular to a kind of method of biological control Phytophthora capsici disease.
Background technology
Phytophthora（Phytophthora）, Peronosporales（Peronosporales）Pathogenic bacteria, low sporangiophore and bacterium
Without significant difference, high has any different silk, and sporangium lemon shape or oval, there is mastoid process on top, is led to the late blight of potato, taro
Epidemic disease etc..For mycelia just without every multinuclear, how at an acute angle branch is, often hangs contracting in bifurcation, thick 2-10 μm, has minority kind that can generate suction
Device forms mycelia and expands body sometimes.Sporangiophore distinguishes less apparent, irregular branch, sympodial branching to differentiation from mycelia, or
Go out from empty sporangium internal layer.Sporangial morphology change greatly, it is generally oval, pyriform, ellipse, also have irregular shape, size
Variation is big；Top has mastoid process, half mastoid process or without mastoid process；General independent basidixed on sporangiophore, once in a while between it is raw；Fall off after maturation or
It does not fall off, the person of falling off has long handle（>20μm）, middle handle（5-20μm）Or short handle（<5μm）；Can direct germination generate germ tube, or indirectly
It sprouts and generates zoospore.Zoospore is oval or fabiform, the double flagellums of side life form cell wall after stopping, spherical, referred to as stops
Only spore.Chlamydospore is mostly spherical shape, thin-walled or heavy wall, colourless to dark, basidixed or life as formed.Oogonium is spherical or close
Spherical, infundibulate, wall is smooth or has decorative pattern, colourless, yellow to brown.Spermary size shape differs, colourless, encloses life（Wear male life）
Or side life.Egg spore is spherical, heavy wall or thin-walled, colourless to light, full device or discontented device.
Capsicum production is influenced by a variety of pest and disease damages, and wherein Phytophthora capsici disease is that main soil passes one of insect pest.Capsicum epidemic disease
Mildew is aggrieved in capsicum time of infertility ground green portion and root, seedling stage fall ill basal part of stem in the water stain shape of dirty-green it is soft rotten or
Volt falls, i.e., basal part of stem when seedling stage falls ill is in dark brown, and seedling withers, and listless dead, wooden You Jing rhizome tissues are rotten, and cauline leaf is anxious
Speed is wilted, and seedling is withered.Strain stem base portion and the aggrieved just less brown spot of generation of branch, then extend rapidly, wrap around
Full stem is included, later stage disease portion is brown, apparent with health tissues boundary line.Root infection diseased plant fibrous root reduce, lateral root be it is filbert or
Dark brown, advanced decay.Stem or root complete stool are withered, and side shoot is aggrieved, and branch is withered thereon.Blade morbidity is in water stain shape by side
Edge is inwardly extended, is filbert afterwards.Floral organ morbidity shows as the soft corruption of browning, falls off.Fruit is mostly fallen ill from the base of a fruit, and initial stage is in water
Stain shape scab, soft corruption when moist, disease is strong to have a common boundary obviously.If environment expands outward suitable for rapid, lead to fruit rot.
Phytophthora capsici disease is caused by Mastigomycotina fungi Phytophthora capsici.Germ is mainly with egg spore, thick Huan's spore in disease
Overwintering wherein soil invalid body bacterial bearing rate is high in residuum or soil and seed, is main dip dyeing source.Condition suitable for when, after overwintering
Germ is splashed through rainwater or irrigation water passes on basal part of stem or plant near the ground, causes to fall ill.Therefore, Phytophthora capsici disease becomes hair
The destructive disease that disease cycle is short, epidemic rate is swift and violent.High temperature, high humidity, rainy days are more, rainfall is big, are conducive to disease.
Control method includes mainly at present：Cultural control, chemical prevention and biological control.Cultural control mainly include rationally the rotation of crops with
And scientic planting.The rationally rotation of crops is tillage control measure very important in agricultural production, rational cropping system and layout, to capsicum
Growth it is highly beneficial.Chemical prevention includes soil medication and field medication；But chemical medication easy tos produce residual and causes ring
Border is polluted, and the generation of Antagonistic Fungi so that the more tired chemopreventive effects the poorer.Biological control mainly uses antagonistic microbe, ring
Border friendly is strong, not will produce pollution.The single microbial inoculum of the prior art is more common, such as streptomycete, bacillus subtilis etc.,
But often there is the defects of control effect is low, drug effect is slow in single bacterial strain, current multiple scientific research institutions have carried out compound life
The research of anti-preparation.Compound biological prevention and control agent generally refer to include two or more microorganisms preparation, select compound biological prevention and control agent
In bacterial strain is more crucial and difficult point；If selection is careless, the reaction of mutual antagonism can be played between bacterial strain instead.It opens
It is our technical issues that need to address to send out a kind of using the method that compound biological prevention and control agent prevents Phytophthora capsici disease.
Invention content
Biological control effect is poor, chemical prevention has the defects of residual in order to overcome in the prior art, and the present invention provides one
The method of kind biological control Phytophthora capsici disease.
The present invention is achieved by the following technical solution：
A kind of method of biological control Phytophthora capsici disease comprising following steps：Biological agent is added to 100-200 times of weight
Water in, then addition, which accounts for, the glucose of 2-3 times of weight of bacteria agent and accounts for the urea of 1-2 times of weight of biological agent, 200rpm
3min is stirred, 28-30 DEG C of activation 3-5h is subsequently placed in, then pepper seedling is immersed into aforesaid liquid 30-60min, takes out capsicum children
Seedling is implanted into soil.
Specifically,
The biological agent includes trichosporon cutaneum, koning trichoderma, bacillus subtilis, Rhodopseudomonas palustris.
Specifically,
The biological agent is prepared according to following technique：
1）Trichosporon cutaneum streak inoculation is cultivated on YPDA culture mediums, obtains single bacterium colony；Picking single bacterium colony is inoculated into YPD liquid
It is cultivated on culture medium, 30 DEG C are cultivated for 24 hours, trichosporon cutaneum seed liquor, for use；
Koning trichoderma streak inoculation is cultivated in PDA culture medium, obtains single bacterium colony；Picking single bacterium colony is inoculated into the training of PDA liquid
It supports and is cultivated on base, 30 DEG C, 200rpm shaking table culture 36h obtain koning trichoderma seed liquor, for use；
First koning trichoderma seed liquor is linked into according to 10% inoculum concentration in fermentation medium, 30 DEG C are cultivated 6 hours, then will
Trichosporon cutaneum seed liquor is linked into fermentation medium according to 5% inoculum concentration, continues to cultivate 18 hours with 30 DEG C, obtains Kang Shi wood
Mould-trichosporon cutaneum mixed fermentation liquid；The group of the fermentation medium is divided into：Corn stalk powder 16g/L, dregs of beans 10g/L, ammonium chloride
5g/L, dipotassium hydrogen phosphate 2g/L, potassium dihydrogen phosphate 1g/L, sodium chloride 0.5g/L, magnesium sulfate 0.1g/L, ferrous sulfate 0.01g/L；
2）By Rhodopseudomonas palustris in the flat lining out cultures of LB, single bacterium colony is obtained；Picking single bacterium colony accesses seed culture medium
To exponential phase, condition of culture is for upper culture：30-32 DEG C, seed liquor is made in illumination 3000-4000Lux；Then according to 10%
Inoculum concentration be linked into fermentation medium, 30-32 DEG C culture for 24 hours, obtain Rhodopseudomonas palustris zymotic fluid；The seed training
The component for supporting base and fermentation medium is：Glucose 8g/L, yeast extract 5g/L, ammonium sulfate 1g/L, potassium dihydrogen phosphate
0.2g/L, dipotassium hydrogen phosphate 0.2g/L, sodium bicarbonate 0.1g/L, magnesium sulfate 0.01g/L；
3）Bacillus subtilis is crossed culture in beef extract-peptone agar medium, obtains single bacterium colony；Picking single bacterium colony
It accesses on seed culture medium, seed liquor is made in 30 DEG C, 200rpm shaking table cultures to exponential phase；Then according to 8% inoculation
Amount is linked into fermentation medium, and 30 DEG C of culture 36h obtain bacillus subtilis fermentation liquor；The seed culture medium and fermentation
The component of culture medium is：Glucose 10g/L, yeast extract 5g/L, ammonium sulfate 3g/L, uremic 1g/L, potassium dihydrogen phosphate 0.1g/L,
Dipotassium hydrogen phosphate 0.1g/L, calcium chloride 0.02g/L, ferrous sulfate 0.01g/L；
4）Koning trichoderma-trichosporon cutaneum mixed fermentation liquid, Rhodopseudomonas palustris zymotic fluid, bacillus subtilis fermentation liquor are pressed
According to 2-3:1-2:The volume ratio of 1-2 is uniformly mixed, then vacuum freeze drying be made bacterium powder to get.
Preferably,
The koning trichoderma is ATCC 66766；The trichosporon cutaneum is ATCC 201110；The Rhodopseudomonas palustris is
ATCC 17001；The bacillus subtilis is ATCC 6633.
Compared with prior art, the advantageous effect that the present invention obtains includes but is not limited to mainly the following aspects：
The present invention prevents Phytophthora capsici using biological agent, residual without environmental pollution caused by chemical prevention and drug
The problems such as staying, is conducive to the No-harmful apple orchard of crop, and peasant can not have to or reduce other prevention phytophthora root rots and improve degeneration-resistant
Property and growth-promoting measure, this can not only reduce the heavy burdens for peasant planting, but also be conducive to improve product quality and yield, increase agriculture
Industry is taken in.
Without antagonism between trichosporon cutaneum and koning trichoderma, can mutual symbiosis, Trichoderma can generate cellulase,
Stalk cellulose is decomposed into the available carbon source of trichosporon cutaneum, so that trichosporon cutaneum utilizes agricultural wastes straw powder point
The carbon source that solution generates is grown, and fermentation costs are reduced；By the way of first accessing koning trichoderma, yeast is accessed after 6 hours
Bacterium；Saccharomycete can also promote the growth of koning trichoderma in turn, and the yield of enzyme of trichoderma is also greatly improved, to improve
The ability of antagonism botrytis cinerea.Rhodopseudomonas palustris and bacillus subtilis can secrete a variety of antibacterial substances, additionally it is possible to disease
Opportunistic pathogen competes nutrition and space；Four kinds of biocontrol bacterial strains are used in complex microorganism preparations of the present invention, and symbiosis, compatibility can be cooperateed with to close
Reason, mutually not antagonism, greatly improves the effect of prevention phytophthora, preparation is to person poultry harmless, environmental-friendly, system harmless to crops
Standby technique is also relatively easy, and application method is convenient, has a extensive future.
Description of the drawings
Fig. 1：Influence of the different biological agents to Phytophthora capsici disease index；
Fig. 2：Influence of the different biological agents to Phytophthora capsici control rate；
Fig. 3：Influence of the different biological agents to capsicum biomass.
Specific implementation mode
In order to make those skilled in the art better understand the technical solutions in the application, having below in conjunction with the application
The technical solution of the application is clearly and completely described in body embodiment, it is clear that described embodiment is only this Shen
Please a part of the embodiment, instead of all the embodiments.Based on the embodiment in the application, those of ordinary skill in the art are not having
There is the every other embodiment obtained under the premise of making creative work, should all belong to the scope of protection of the invention.
Embodiment 1
A kind of method of biological control Phytophthora capsici disease comprising following steps：Biological agent is added to the water of 100 times of weight
In, then addition accounts for the glucose of 2 times of weight of bacteria agent, accounts for the urea of 1 times of weight of biological agent, and 200rpm stirs 3min,
30 DEG C of activation 3h are subsequently placed in, then pepper seedling is immersed into aforesaid liquid 30min, pepper seedling is taken out, is implanted into soil.
The biological agent includes trichosporon cutaneum, koning trichoderma, bacillus subtilis, Rhodopseudomonas palustris.
Specifically, the biological agent is prepared according to following technique：
Trichosporon cutaneum streak inoculation is cultivated on YPDA culture mediums, obtains single bacterium colony；Picking single bacterium colony is inoculated into the training of YPD liquid
It supports and is cultivated on base, 30 DEG C of cultures for 24 hours, obtain seed liquor, for use；
The formula of YPD culture mediums：Glucose 20g, peptone 20g, yeast extraction leaching powder 10g, supplement distilled water to 1000mL；
The formula of YPDA culture mediums：Glucose 20g, peptone 20g, yeast extraction leaching powder 10g, agar 20g, supplement distilled water
To 1000mL；
Koning trichoderma streak inoculation is cultivated in PDA culture medium, obtains single bacterium colony；Picking single bacterium colony is inoculated into the training of PDA liquid
It supports and is cultivated on base, 30 DEG C, 200rpm shaking table culture 36h obtain seed liquor, for use；
First by koning trichoderma seed liquor according to 10% inoculum concentration（Inoculum density is 1 × 107CFU/ml）It is linked into fermentation medium
In, 30 DEG C cultivate 6 hours, then by trichosporon cutaneum seed liquor according to 5% inoculum concentration（Inoculum density is 1 × 107CFU/ml）It connects
Enter to fermentation medium, continues to cultivate 18 hours with 30 DEG C, obtain koning trichoderma-trichosporon cutaneum mixed fermentation liquid；The fermentation
The group of culture medium is divided into：Corn stalk powder 16g/L, dregs of beans 10g/L, ammonium chloride 5g/L, dipotassium hydrogen phosphate 2g/L, potassium dihydrogen phosphate
1g/L, sodium chloride 0.5g/L, magnesium sulfate 0.1g/L, ferrous sulfate 0.01g/L；
By Rhodopseudomonas palustris in the flat lining out cultures of LB, single bacterium colony is obtained；Picking single bacterium colony accesses on seed culture medium
To exponential phase, condition of culture is for culture：30-32 DEG C, seed liquor is made in illumination 3000-4000Lux；Then according to 10%
Inoculum concentration（Inoculum density is 3 × 107CFU/ml）It is linked into fermentation medium, 30-32 DEG C of culture for 24 hours, obtains the red vacation in marsh
Unit cell fermented liquid；The component of the seed culture medium and fermentation medium is：Glucose 8g/L, yeast extract 5g/L,
Ammonium sulfate 1g/L, potassium dihydrogen phosphate 0.2g/L, dipotassium hydrogen phosphate 0.2g/L, sodium bicarbonate 0.1g/L, magnesium sulfate 0.01g/L；
Bacillus subtilis is crossed culture in beef extract-peptone agar medium, obtains single bacterium colony；Picking single bacterium colony connects
Enter on seed culture medium, seed liquor is made in 30 DEG C, 200rpm shaking table cultures to exponential phase；Then according to 8% inoculum concentration
（Inoculum density is 5 × 106CFU/ml）It is linked into fermentation medium, 30 DEG C of culture 36h obtain fermentation of bacillus subtilis
Liquid；The component of the seed culture medium and fermentation medium is：Glucose 10g/L, yeast extract 5g/L, ammonium sulfate 3g/L, urine
Malicious 1g/L, potassium dihydrogen phosphate 0.1g/L, dipotassium hydrogen phosphate 0.1g/L, calcium chloride 0.02g/L, ferrous sulfate 0.01g/L；
By koning trichoderma-trichosporon cutaneum mixed fermentation liquid, Rhodopseudomonas palustris zymotic fluid, bacillus subtilis fermentation liquor according to
2:1:1 volume ratio is uniformly mixed, then vacuum freeze drying be made bacterium powder to get.
The koning trichoderma is ATCC 66766；The trichosporon cutaneum is ATCC 201110；The Rhodopseudomonas palustris
For ATCC 17001；The bacillus subtilis is ATCC 6633.
Embodiment 2
A kind of method of biological control Phytophthora capsici disease comprising following steps：Biological agent is added to the water of 200 times of weight
In, then addition accounts for the glucose of 3 times of weight of bacteria agent, accounts for the urea of 2 times of weight of biological agent, and 200rpm stirs 3min,
28 DEG C of activation 5h are subsequently placed in, then pepper seedling is immersed into aforesaid liquid 60min, pepper seedling is taken out, is implanted into soil.
The biological agent includes trichosporon cutaneum, koning trichoderma, bacillus subtilis, Rhodopseudomonas palustris.
Specifically, the biological agent is prepared according to following technique：
Trichosporon cutaneum streak inoculation is cultivated on YPDA culture mediums, obtains single bacterium colony；Picking single bacterium colony is inoculated into the training of YPD liquid
It supports and is cultivated on base, 30 DEG C of cultures for 24 hours, obtain seed liquor, for use；
The formula of YPD culture mediums：Glucose 20g, peptone 20g, yeast extraction leaching powder 10g, supplement distilled water to 1000mL；
The formula of YPDA culture mediums：Glucose 20g, peptone 20g, yeast extraction leaching powder 10g, agar 20g, supplement distilled water
To 1000mL；
Koning trichoderma streak inoculation is cultivated in PDA culture medium, obtains single bacterium colony；Picking single bacterium colony is inoculated into the training of PDA liquid
It supports and is cultivated on base, 30 DEG C, 200rpm shaking table culture 36h obtain seed liquor, for use；
First by koning trichoderma seed liquor according to 10% inoculum concentration（Inoculum density is 1 × 107CFU/ml）It is linked into fermentation medium
In, 30 DEG C cultivate 6 hours, then by trichosporon cutaneum seed liquor according to 5% inoculum concentration（Inoculum density is 1 × 107CFU/ml）It connects
Enter to fermentation medium, continues to cultivate 18 hours with 30 DEG C, obtain koning trichoderma-trichosporon cutaneum mixed fermentation liquid；The fermentation
The group of culture medium is divided into：Corn stalk powder 16g/L, dregs of beans 10g/L, ammonium chloride 5g/L, dipotassium hydrogen phosphate 2g/L, potassium dihydrogen phosphate
1g/L, sodium chloride 0.5g/L, magnesium sulfate 0.1g/L, ferrous sulfate 0.01g/L；
By Rhodopseudomonas palustris in the flat lining out cultures of LB, single bacterium colony is obtained；Picking single bacterium colony accesses on seed culture medium
To exponential phase, condition of culture is for culture：30-32 DEG C, seed liquor is made in illumination 3000-4000Lux；Then according to 10%
Inoculum concentration（Inoculum density is 3 × 107CFU/ml）It is linked into fermentation medium, 30-32 DEG C of culture for 24 hours, obtains the red vacation in marsh
Unit cell fermented liquid；The component of the seed culture medium and fermentation medium is：Glucose 8g/L, yeast extract 5g/L,
Ammonium sulfate 1g/L, potassium dihydrogen phosphate 0.2g/L, dipotassium hydrogen phosphate 0.2g/L, sodium bicarbonate 0.1g/L, magnesium sulfate 0.01g/L；
Bacillus subtilis is crossed culture in beef extract-peptone agar medium, obtains single bacterium colony；Picking single bacterium colony connects
Enter on seed culture medium, seed liquor is made in 30 DEG C, 200rpm shaking table cultures to exponential phase；Then according to 8% inoculum concentration
（Inoculum density is 5 × 106CFU/ml）It is linked into fermentation medium, 30 DEG C of culture 36h obtain fermentation of bacillus subtilis
Liquid；The component of the seed culture medium and fermentation medium is：Glucose 10g/L, yeast extract 5g/L, ammonium sulfate 3g/L, urine
Malicious 1g/L, potassium dihydrogen phosphate 0.1g/L, dipotassium hydrogen phosphate 0.1g/L, calcium chloride 0.02g/L, ferrous sulfate 0.01g/L；
By koning trichoderma-trichosporon cutaneum mixed fermentation liquid, Rhodopseudomonas palustris zymotic fluid, bacillus subtilis fermentation liquor according to
3:2:2 volume ratio is uniformly mixed, then vacuum freeze drying be made bacterium powder to get.
The koning trichoderma is ATCC 66766；The trichosporon cutaneum is ATCC 201110；The Rhodopseudomonas palustris
For ATCC 17001；The bacillus subtilis is ATCC 6633.
Embodiment 3
Control effect of the present invention to P. capsici
Biological agent group：Experimental group：Embodiment 1；Control group 1：Only with koning trichoderma-trichosporon cutaneum mixed fermentation liquid,
Remaining is the same as embodiment 1；Control group 2：Only with Rhodopseudomonas palustris zymotic fluid and bacillus subtilis fermentation liquor, remaining is same
Embodiment 1；Control group 3：Koning trichoderma and trichosporon cutaneum are separately fermented, then four kinds of zymotic fluids are mixed, remaining is the same as real
Apply example 1；Blank control：The clear water of isodose.
Mode of operation is the same as embodiment 1.
Soil pretreatment：Phytophthora blight of pepper accesses after solid culture 5 days in V8 fluid nutrient mediums, and 25 DEG C are cultivated 5 days,
Fluid nutrient medium is poured out, mycelia is weighed, and is linked into each cell soil by the amount of 8g/ kilograms of soil.
Processing mode：Capsicum experimental plot, is divided into five cells, and each cell corresponds to each group；Pepper seedling is pressed respectively
According to the mode of embodiment 1（Biological agent is different）After processing, it is transplanted to each cell；Sampling statistics occurring degree, calculates after 21 days
Disease index；Also have detected the total fresh weight of plant and root fresh weight.
Examination criteria：Phytophthora root rot occurring degree is divided into 6 grades.0 grade is not fallen ill for root system；1 grade is root system incidence≤30%,
Blade is normal；2 grades are 30%<Root system incidence≤60%, blade are normal；3 grades are 60%<Root system incidence≤80%；Blade
Turn yellow, 4 grades are 80% or more root system incidence, and blade is withered, and 5 grades dead for whole strain, and blade is withered.Disease index and prevention are imitated
Fruit calculates according to the following formula respectively:Disease index=[(sick grade strain number × typical value)/(total strain number × highest disease grade represents
Value)] × 100.Control rate=[(control disease index-processing disease index)/control disease index] × 100%.
As shown in Figs. 1-2, control rate highest of the experimental group to Phytophthora capsici, hence it is evident that be higher than control group 1-2, also above separately
The control group 3 of fermentation method illustrates that Trichoderma can be accessed first with co-fermentation between trichosporon cutaneum and koning trichoderma, produces
Cellulose decomposition is the available carbon source of trichosporon cutaneum by raw cellulase, so that the trichosporon cutaneum of subsequent inoculations utilizes
The carbon source that agricultural wastes straw powder generates is grown, and fermentation costs are reduced, and saccharomycete can also promote Kang Shi wooden in turn
Mould growth, and the yield of enzyme of trichoderma is also greatly improved, to improve the ability of antagonism botrytis cinerea.Plant is to lean on root system
It is drawn from soil and utilizes mineral nutrient, maintain itself growth and breeding, root system is more flourishing, and plant growth is better；Plant generation
The biomass of table plant, can be as the important indicator of assessment fruit yield and quality；As shown in figure 3, experimental group, control group 1-
3 biomass is apparently higher than blank control group, wherein the total fresh weight of plant and root fresh weight two indices is selected, obviously higher than sky
White control, the weightening of wherein experimental group biomass are the most notable.
Although above having used general explanation, specific implementation mode and experiment, the present invention is described in detail,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (4)
1. a kind of method of biological control Phytophthora capsici disease comprising following steps：Biological agent is added to 100-200 times to weigh
In the water of amount, then addition accounts for the glucose of 2-3 times of weight of bacteria agent and accounts for the urea of 1-2 times of weight of biological agent,
200rpm stirs 3min, is subsequently placed in 28-30 DEG C of activation 3-5h, then pepper seedling is immersed aforesaid liquid 30-60min, takes out
Pepper seedling is implanted into soil.
2. according to the method described in claim 1, it is characterized in that, the biological agent include trichosporon cutaneum, it is koning trichoderma, withered
Careless bacillus, Rhodopseudomonas palustris.
3. method according to claim 1 or 2, which is characterized in that the biological agent is prepared according to following technique：
1）Trichosporon cutaneum streak inoculation is cultivated on YPDA culture mediums, obtains single bacterium colony；Picking single bacterium colony is inoculated into YPD liquid
It is cultivated on culture medium, 30 DEG C are cultivated for 24 hours, trichosporon cutaneum seed liquor, for use；
Koning trichoderma streak inoculation is cultivated in PDA culture medium, obtains single bacterium colony；Picking single bacterium colony is inoculated into the training of PDA liquid
It supports and is cultivated on base, 30 DEG C, 200rpm shaking table culture 36h obtain koning trichoderma seed liquor, for use；
First koning trichoderma seed liquor is linked into according to 10% inoculum concentration in fermentation medium, 30 DEG C are cultivated 6 hours, then will
Trichosporon cutaneum seed liquor is linked into fermentation medium according to 5% inoculum concentration, continues to cultivate 18 hours with 30 DEG C, obtains Kang Shi wood
Mould-trichosporon cutaneum mixed fermentation liquid；The group of the fermentation medium is divided into：Corn stalk powder 16g/L, dregs of beans 10g/L, ammonium chloride
5g/L, dipotassium hydrogen phosphate 2g/L, potassium dihydrogen phosphate 1g/L, sodium chloride 0.5g/L, magnesium sulfate 0.1g/L, ferrous sulfate 0.01g/L；
2）By Rhodopseudomonas palustris in the flat lining out cultures of LB, single bacterium colony is obtained；Picking single bacterium colony accesses seed culture medium
To exponential phase, condition of culture is for upper culture：30-32 DEG C, seed liquor is made in illumination 3000-4000Lux；Then according to 10%
Inoculum concentration be linked into fermentation medium, 30-32 DEG C culture for 24 hours, obtain Rhodopseudomonas palustris zymotic fluid；The seed training
The component for supporting base and fermentation medium is：Glucose 8g/L, yeast extract 5g/L, ammonium sulfate 1g/L, potassium dihydrogen phosphate
0.2g/L, dipotassium hydrogen phosphate 0.2g/L, sodium bicarbonate 0.1g/L, magnesium sulfate 0.01g/L；
3）Bacillus subtilis is crossed culture in beef extract-peptone agar medium, obtains single bacterium colony；Picking single bacterium colony
It accesses on seed culture medium, seed liquor is made in 30 DEG C, 200rpm shaking table cultures to exponential phase；Then according to 8% inoculation
Amount is linked into fermentation medium, and 30 DEG C of culture 36h obtain bacillus subtilis fermentation liquor；The seed culture medium and fermentation
The component of culture medium is：Glucose 10g/L, yeast extract 5g/L, ammonium sulfate 3g/L, uremic 1g/L, potassium dihydrogen phosphate 0.1g/L,
Dipotassium hydrogen phosphate 0.1g/L, calcium chloride 0.02g/L, ferrous sulfate 0.01g/L；
4）Koning trichoderma-trichosporon cutaneum mixed fermentation liquid, Rhodopseudomonas palustris zymotic fluid, bacillus subtilis fermentation liquor are pressed
According to 2-3:1-2:The volume ratio of 1-2 is uniformly mixed, then vacuum freeze drying be made bacterium powder to get.
4. according to the method described in claim 2-3, which is characterized in that the koning trichoderma is ATCC 66766；The silk spore
Yeast is ATCC 201110；The Rhodopseudomonas palustris is ATCC 17001；The bacillus subtilis is ATCC 6633.
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