CN106305793A - Compound bacterial manure used for prevention and control of cotton verticillium wilt and the preparation method thereof - Google Patents
Compound bacterial manure used for prevention and control of cotton verticillium wilt and the preparation method thereof Download PDFInfo
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Abstract
The invention discloses a compound bacterial manure used for prevention and control of cotton verticillium wilt and the preparation method thereof. Materials of the compound bacterial manure consist of Streptomyces aburaviensis, Streptomyces tanashiensis, Brevibacillus agri, Bacillus clausii, Trichoderma citrinoviride, and potassium fulvic acid. Bacteria of the invention can make full use of the interspecific or intraspecific antibiosis, competition, hyperparasite, and bacteriolysis of microbes, and induce cotton to generate the disease resistance ability through secondary metabolite, thereby producing various antibiotic substances such as luteomycin and aburamycin to prevent the pathogenic bacteria of cotton verticillium wilt. The 5 strains used in the invention can be obtained through separation from soil directly; the strains can promote the growth of root, and secrete some secondary metabolites to plant roots while growing on the plant surface, inside the plant or in soil, thereby improving the plant's absorption of nutrients, and promoting the plant growth like fertilizers.
Description
Technical field
The invention belongs to Strategies of Agricultural Bio-control and microbial manure technical field, be specifically related to a kind of preventing and treating cotton verticillium wilt
Composite microbic bacterial fertilizer and preparation method thereof.
Background technology
Cotton verticillium wilt is that the harm caused by Dahlia Pinnata Cav. Verticillium (Verticillium dahaliae) is serious and difficult
Soil-borne disease with preventing and treating.Cotton verticillium wilt is at China's population outbreak regularly, the sown areas of cotton being affected by every year
More than 40,000,000 mu, causing the Cotton Gossypii underproduction 10 about %-20%, disease occurs serious plot to cause the underproduction may be up to throughout the year
60%-70%。
The method of preventing and treating cotton verticillium wilt mainly has: one is chemical prevention, but chemopreventive effects is poor, and easily
Cause environmental pollution, destroy the ecological balance in farmland；Two is crop rotation, but under the conditions of the most specific agricultural environment, people is manyly
Less and substitute Cotton Gossypii high price crop species doctor weary, it is difficult to popularization and application；Three is Biological control, and Biological control is owing to having environment
Pollute little, to person poultry safety, specialization is strong, be not likely to produce Drug resistance, effect lasting period length, can effectively Protect natural enemies, produce into
The advantages such as this is low are increasingly subject to the favor of people.
The probiotics being presently used for preventing and treating crop verticillium wilt mainly has bacillus cereus, trichoderma, actinomycetes etc., achieves
Significantly prevention effect.But owing to multiformity and the synchronization of pathogen are evolved, the biocontrol effect of single bacterial strain is the most unstable,
DeGrain.
Bacillus cereus can produce multiple Substance, simultaneously as bacillus cereus is present in soil, is that soil passes
Disease control is studied awfully hot region.In terms of preventing and treating cotton verticillium wilt, what the bacillus cereus for controlling experiment had goes back
Can survive in cotton plant body and surely grow.Bacillus amyloliquefaciens HMB-1005 bacterial strain can be at Cotton Gossypii Colonization inside plants, basin
Plant test and prove that it has certain resistance to cotton verticillium wilt, meanwhile, this bacterial strain can also long in soil time
Between
Holding (Guo Huijuan etc. 2011).Bacillus polymyxa 7-4 bacterial strain is utilized lysoplate assay and screens, and presses down
Bacterium activity higher (Wang Xiaoying etc. 2011).The highest preventive effect of domestic current Biological control verticillium wilt is bacillus subtilis C-02
Bacterial strain, potted plant preventive effect reaches 85.4%, and its antibiotic substance is also separated, it was demonstrated that be a kind of antibacterial protein or peptides (Nie Taili etc. 2011
).In Cotton Gossypii body, the bacillus subtilis BSD-2 bacterial strain of isolated can significantly inhibit verticillium dahliae spore germination, its
Activated product is own through being separated, and has good heat stability and acid and alkali-resistance character, has very much application prospect (Zhang Duo etc. 2008
).The bacillus cereus for Biological control raw in Cotton Gossypii also has Paenibacillus polymyxa (Paenibacillus polymyxa)
Jsas cd bacterial strain, this bacterial strain can apply to the root irrigation of cotton Seedling, and field district test proves that using this bacterial strain also has simultaneously
Help the raising (Lin Ling etc. 2010) of output of cotton.
Trichoderma spp. is a kind of saprophytic fungus, has the strongest adaptation ability, on soil, plant rhizosphere and other casts very
Easily growth and breeding.Owing to Trichoderma spp. is widely distributed, obtaining so can be easily separated, the research to Trichoderma spp. is also compared many.At present, entirely
Oneself is through there being more than 50 kind of Trichoderma preparation listing in the world, is used for preventing and treating plurality of plant diseases and achieving gratifying effect.Trichoderma spp.
Prevent and treat the same achievement of the research in terms of verticillium wilt rich.Trichoderma spp. can effectively prevent and treat causing harm of verticillium dahliae, the former Russian scholar
Find that its preventive effect is up to more than 40%.The research of trichoderma is also compared many by China, trichoderma and verticillium dahliae at culture dish
Interior opposite culture, finds that Trichoderma spp. can significantly inhibit the growth of pathogen, and microexamination is it appeared that the mycelia of verticillium dahliae
Fracture, dissolving and protoplasm concentrate.Owing to cotton verticillium wilt has twice onset peak, investigate field diseases card when onset peak
Bright, Trichoderma spp. T34, THT and T24 respectively reach 38.2%, 53.5% and 13.6% to the preventive effect of cotton verticillium wilt, and 36%.Breathing out thatch wood
Mould and alkene acyl beautiful jade is made water dispersible granules and is found, in pot experiment, the preventive effect to verticillium wilt reaches as high as 88%, and will not shadow
Ring the normal growth of cotton plants.In biocontrol fungi, trichoderma research is a lot, and its mechanism of action also comparative maturity is generally acknowledged at present
Have antagonism, superparasitism, competition and induction crop resistance etc..
Actinomycetes mainly live in earth's surface, can produce Multiple Classes of Antibiotics, are referred to as " antibiotic factory ".From the angle of antagonism
Degree sets out, and selects actinomycetes verticillium wilt to carry out controlling experiment for finding efficient biological and ecological methods to prevent plant disease, pests, and erosion method significant,
It is true that a lot of scholars have done trial in this respect and have achieved a series of achievement.These trials are carried out around streptomycete, typically
Utilize flat board face-off method screening Antagonistic Fungi, further investigate again after then measuring its preventive effect.Pot experiment is had to prove, actinomycetes
Be combined the generation that can efficiently prevent and treat verticillium wilt with fertilizer, potted plant preventive effect reaches 57%.Streptomycete S23 has stronger suppression
The effect of verticillium dahliae, in addition, China scientific research personnel also the S5 contour active bacterial strain from different regions isolated, basin
Plant in test and all show certain preventive effect.
Due to multiformity and the synchronization evolution characteristic of pathogen, single antagonistic strain is also unable to reach highly desirable preventing and treating effect
Really, therefore, a lot of biological functions of biocontrol microorganisms need the synergism relied between the antibacterial of more than two strains or two strains, competence exertion
Go out preferably effect.
Summary of the invention
The first object of the present invention is to provide a kind of composite microbic bacterial fertilizer preventing and treating cotton verticillium wilt.This composite microbial
The proportioning raw materials of thing bacterial manure is: A Bula streptomycete mycopowder 10-30 part, and field is without streptomycete mycopowder 10-30 part, soil short spore bar
Bacterium mycopowder 10-30 part, Bacillus clausii mycopowder 10-30 part, Fructus Citri tangerinae green trichoderma mycopowder 10-30 part, potassium fulvate 10-30 part.
In described microbial-bacterial fertilizer, A Bula streptomycete, field is without streptomycete, soil bacillus brevis, Bacillus clausii, and Fructus Citri tangerinae is green
Trichoderma spp., five kinds of bacterium are the energy symbiotic co-existences that screening obtains from the microbial strains of strain more than 450, can produce multiple antibiotics such as Resina garciniae
Mycin and aburamycin；To suppression, cotton verticillium wilt pathogen--Dahlia Pinnata Cav. Verticillium has the most powerful effect for they；Separately
On the one hand, the strain of the present invention can comprehensively utilize the antibiosis between microbial species or in kind, competition, superparasitism, bacteriolysis,
Or produce disease resistance by secondary metabolite inducing cotton, strengthen its protection effect；Again, this used in the present invention
5 strain bacterial strains are all can be directly isolated to obtain from soil, have fixed nitrogen and root mark growth-promoting functions, can be in crop surface, plant
In portion or soil while flourish, and orient plant rhizosphere and secrete some secondary metabolite, it is possible to increase plant is to supporting
Point absorption, stimulate plant strain growth to play the effect of fertilizer；
The second object of the present invention is to provide the preparation method of a kind of composite microbic bacterial fertilizer preventing and treating cotton verticillium wilt, the party
Method step is simple, it is ensured that the medicine fertilizer of microbial-bacterial fertilizer is remarkably productive.
In order to achieve the above object, the preparation method of the composite microbic bacterial fertilizer of this preventing and treating cotton verticillium wilt, including following
Step:
Step one, the preparation of A Bula streptomycete mycopowder
Take out A Bula streptomyces species preservation pipe, draw flat board with Gause I solid medium and recover, cultivate 7 for 30 DEG C
My god.Under flat board, the streak inoculation of picking single bacterium colony is equipped with in the Fructus Solani melongenae bottle of 150 milliliters of Gause I solid mediums, is cultivating
Case is cultivated 6-8 days for 30 DEG C, treats that lawn covers with Fructus Solani melongenae bottle, produce a large amount of spore powder and i.e. can use the physiological saline solution of 500 milliliters
Eluting, regulation spore concentration is 0.1 hundred million cfu/ml, is A Bula streptomycete seed liquor；
Secondary seed spreads cultivation: the A Bula streptomycete seed liquor of above-mentioned preparation is seeded to the A Bula of sterilizing with the inoculum concentration of 1%
On streptomycete liquid two stage seed culture medium, the fermentation tank liquid amount of 500L is 350L, ventilation be liquid-gas ratio per minute be 1:
Cultivate 36-48 hour, when thalline content reaches 80g/L, can be used as A Bula streptomycete secondary seed for 0.8,28-30 DEG C
Liquid；
Fermentation: the A Bula streptomycete secondary seed solution of above-mentioned preparation is seeded to the inoculum concentration of 20% Ah cloth's slide fastener of sterilizing
In mould bacteria solid fermentation culture medium, after strain is mixed homogeneously with A Bula streptomycete solid medium, the A Bula planted will be connected
Streptomycete solid fermentation culture medium is spread out on fermentation tank, and height of materials is 5-10cm, temperature control 28-32 DEG C, a few days ago air humidity
Remaining 60%-75%, air humidity remains 40%-50% later, ferments 3-5 days, detects its spore content and is not less than 3,000,000,000
CFU/g, gets final product low-temperature air-drying, treats that moisture is less than 10%, pulverizes 100 mesh sieves, i.e. obtains perfectly round streptomycete mycopowder；
Wherein, described Gause I solid medium: potassium nitrate: 1 gram, soluble starch: 20 grams, dipotassium hydrogen phosphate: 05,
Magnesium sulfate: 05, sodium chloride: 05, ferrous sulfate: 0 01 grams, agar: 20 grams, 1000ml, PH7 2～7 4 supplied by distilled water；
Wherein, described A Bula streptomycete secondary liquid seed culture medium: rapeseed cake powder 2%, tapioca starch 2%, sodium chloride 0.2%, carbon
Acid calcium 1%, magnesium sulfate 0.1%, PH7 2～7 4；
Wherein, described A Bula streptomycete solid fermentation culture medium: thick skin of Semen Maydis 80%, rapeseed cake 2%, tapioca starch 1%, stone powder 9%,
Wheat bran 8%, manganese sulfate 0.004%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate 0.05%, solid medium water content is 40%-60%.Each training
The condition supporting base sterilizing is: 0.10-0.15MPa, 121 DEG C of sterilizings 30 minutes.
Step 2, field is without the preparation of streptomycete mycopowder
Taking-up field, without streptomyces species preservation pipe, is drawn flat board with Gause I solid medium and is recovered, and cultivates 7 days for 30 DEG C.
Under flat board, the streak inoculation of picking single bacterium colony is equipped with in the Fructus Solani melongenae bottle of 150 milliliters of Gause I solid mediums, at incubator
In 30 DEG C cultivate 6-8 days, treat that lawn covers with Fructus Solani melongenae bottle, produce a large amount of spore powder and i.e. can use the physiological saline solution of 500 milliliters to wash
De-, regulation spore concentration is 0.1 hundred million cfu/ml, is field without streptomycete seed liquor；
Secondary seed spreads cultivation: the field of above-mentioned preparation is seeded to the field of sterilizing without strepto-without streptomycete seed liquor with the inoculum concentration of 1%
In bacteria liquid secondary seed medium, the fermentation tank liquid amount of 500L is 350L, ventilation be liquid-gas ratio per minute be 1:0.8,
Cultivate 36-48 hour, when thalline content reaches 60g/L, can be used as field without streptomycete secondary seed solution for 28-30 DEG C；
Fermentation: the field of above-mentioned preparation is seeded to the field of sterilizing without streptomycete without streptomycete secondary seed solution with the inoculum concentration of 20%
In solid fermentation culture medium, after strain is mixed homogeneously without streptomycete solid medium with field, solid without streptomycete by connecting the field planted
Body fermentation medium is spread out on fermentation tank, and height of materials is 5-10cm, and temperature control 28-32 DEG C, a few days ago air humidity remains
60%-75%, air humidity remains 40%-50% later, ferments 3-5 days, detects its spore content and is not less than 5,000,000,000 CFU/g, i.e.
Can low-temperature air-drying, treat that moisture is less than 10%, pulverized 100 mesh sieves, i.e. obtain field without streptomycete mycopowder；
Wherein, described Gause I solid medium: potassium nitrate: 1 gram, soluble starch: 20 grams, dipotassium hydrogen phosphate: 05,
Magnesium sulfate: 05, sodium chloride: 05, ferrous sulfate: 0 01 grams, agar: 20 grams, 1000ml, PH7 2～7 4 supplied by distilled water；
Wherein, described field is without streptomycete secondary liquid seed culture medium: cottonseed meal powder 2%, Semen Maydis powder 2%, sodium chloride 0.2%, carbonic acid
Calcium 1%, magnesium sulfate 0.1%, dipotassium hydrogen phosphate 0.1%, PH7 2～7 4；
Wherein, described field is without streptomycete solid fermentation culture medium: bagasse 80%, rapeseed cake 2%, tapioca starch 1%, stone powder 9%, slightly beautiful
Silverskin 8%, manganese sulfate 0.004%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate 0.05%, solid medium water content is 40%-60%.Each training
The condition supporting base sterilizing is: 0.10-0.15MPa, 121 DEG C of sterilizings 30 minutes.
Step 3, the preparation of soil bacillus brevis mycopowder
Taking out soil bacillus brevis preservation pipe, draw flat board respectively with nitrogen-free solid medium and recover, 30 DEG C of cultivations 72 are little
Time.Under flat board, the single colony inoculation of picking is to equipped with nitrogen-free solid medium, cultivates 72 hours in 30 DEG C of incubators, uses
3000ml sterilized water, by the lawn eluting in three Fructus Solani melongenae bottles, is seeded to equipped with 300L soil bacillus brevis seed culture medium
In 500L fermentation tank, opening stirring 120r/min, within first 8 hours, ventilation is 100L/min, and after 8-24 hour, ventilation is 200L/
Min, after 24 hours, ventilation is 300L/min, cultivates 32-48 hour, treats that total bacteria count content is not less than 2,000,000,000 cfu/ for 30 DEG C
Ml, can be used as soil bacillus brevis seed liquor；
Fermentation: the soil bacillus brevis seed liquor of above-mentioned preparation is seeded to soil bacillus brevis with the ratio of 10%-20%
On solid medium, mixing, in tray top fermentation, windrow thickness is 5-10cm, controls fermentation temperature and is 30-40 DEG C, and ferment 36-
48 hours, treat that spore content is not less than 5,000,000,000 cfu/g, can dry, treat that moisture is less than 10%, pulverized 100 mesh sieves, i.e.
Obtain soil bacillus brevis mycopowder；
Wherein, described nitrogen-free solid medium: sucrose 10g, dipotassium hydrogen phosphate 0.2g, magnesium sulfate 0.2g, sodium chloride 0.2g, sulphuric acid
Calcium 0.1g, calcium carbonate 5g, agar 18g, distilled water 1000mL, pH7.2；
Wherein, described soil bacillus brevis seed culture medium: molasses 20g/L, corn starch 10 g/L, cottonseed meal powder 40 g/L,
Magnesium sulfate 0.4 g/L, manganese sulfate 0.6g/L, potassium dihydrogen phosphate 0.6 g/L, calcium carbonate 10 g/L, pH7.0.Each medium sterilization
Condition be: 0.10-0.15MPa, 121 DEG C of sterilizings 30 minutes；
Wherein, described soil bacillus brevis solid medium: bagasse 57%, wheat bran 35%, cottonseed meal 5%, stone powder 1%, slightly beautiful
Silverskin 2%, carbamide 0.4%, dipotassium hydrogen phosphate 0.4%, magnesium sulfate 0.05%, moisture content in medium 50%-60%.Each medium sterilization
Condition is: 0.10-0.15MPa, 121 DEG C of sterilizings 30 minutes.
Step 4, the preparation of Bacillus clausii mycopowder
Take out Bacillus clausii preservation pipe, draw flat board respectively with nutrient solid medium and recover, 30 DEG C of cultivations
48 hours.Under flat board, the single colony inoculation of picking is to equipped with nutrient solid medium, cultivates 48 in 30 DEG C of incubators
Hour, with 3000ml sterilized water by the lawn eluting in three Fructus Solani melongenae bottles, it is seeded to equipped with 300L liquid seed culture medium
In 500L fermentation tank, opening stirring 120r/min, within first 10 hours, ventilation is 200L/min, and after 10 hours, ventilation is 320L/min
, cultivate 16-24 hour, treat that total bacterial content is not less than 2,000,000,000 cfu/ml, can be used as Bacillus clausii seed liquor for 30 DEG C；
Fermentation: the Bacillus clausii seed liquor of above-mentioned preparation is added to Ke Lao with the inoculum concentration of the 10%-20% of total inoculum concentration
In family name's bacillus cereus solid fermentation culture medium, mixing, in tray top fermentation, windrow thickness is 5-10cm, controls fermentation temperature and is
30-40 DEG C, ferment 36-48 hour, treat that spore content is not less than 8,000,000,000 cfu/g, can dry, treat that moisture is less than 10%, powder
Broken mistake 100 mesh sieve, i.e. obtains Bacillus clausii mycopowder；
Wherein, described nutrient broth solid medium: peptone 10.0 g/L, Carnis Bovis seu Bubali cream 3.0 g/L, sodium chloride 5.0
G/L, agar 20 g/L, pH 7.2 ± 0.2；
Wherein, described liquid seed culture medium: molasses 35 g/L, bean cake powder 20 g/L, magnesium sulfate 0.6g/L, manganese sulfate 0.5
G/L, potassium dihydrogen phosphate 0.3 g/L, calcium carbonate 5.0 g/L, pH7.0；
Wherein, Bacillus clausii solid fermentation culture medium: wheat bran 50%, thick skin of Semen Maydis 42%, cottonseed meal 5%, stone powder 1%, medicated beer
Grain 2%, carbamide 0.4%, dipotassium hydrogen phosphate 0.4%, magnesium sulfate 0.05%, moisture content in medium 50%-60%.The bar of each medium sterilization
Part is: 0.10-0.15MPa, 121 DEG C of sterilizings 30 minutes.
Step 5, the preparation of Fructus Citri tangerinae green trichoderma mycopowder
A, the preparation of Fructus Citri tangerinae green trichoderma seed liquor: take out Fructus Citri tangerinae green trichoderma culture presevation pipe, draw flat board with PDA solid medium and carry out multiple
Soviet Union, cultivates 6 days for 30 DEG C.Under flat board, the streak inoculation of picking single bacterium colony is equipped with the Fructus Solani melongenae bottle of 150 milliliters of PDA solid mediums
In, cultivate 4-6 days for 30 DEG C in incubator, treat that lawn covers with Fructus Solani melongenae bottle, produce a large amount of spore and i.e. can use the aseptic life of 500 milliliters
Reason salt water, regulation spore concentration is 0.1 hundred million cfu/ml, is Fructus Citri tangerinae green trichoderma seed liquor；
Fermentation: the Fructus Citri tangerinae green trichoderma seed liquor of above-mentioned preparation is seeded to the inoculum concentration of 2% the Fructus Citri tangerinae green trichoderma solid fermentation training of sterilizing
Support on base, after strain is mixed homogeneously with Fructus Citri tangerinae green trichoderma solid medium, the Fructus Citri tangerinae green trichoderma solid fermentation culture medium stand planted will be connected
On fermentation tank, height of materials is 5-10cm, temperature control 28-32 DEG C, and humidity remains 60%-75%, ferments 4-6 days, detects it total
Spore content is not less than 3,000,000,000 CFU/g, gets final product low-temperature air-drying, treats that moisture is less than 10%, pulverized 100 mesh sieves, and i.e. obtained Fructus Citri tangerinae
Green trichoderma mycopowder；
Wherein, described PDA solid medium: Rhizoma Solani tuber osi 200g, sucrose 20g, water 1000mL, agar 20g；
Wherein, described Fructus Citri tangerinae green trichoderma solid fermentation culture medium: bagasse 85%, the sesame residues dregs of rice 2%, corn cob 5%, stone powder 4%, slightly
Skin of Semen Maydis 2%, carbamide 0.4%, dipotassium hydrogen phosphate 0.4%, magnesium sulfate 0.05%, moisture content in medium 50%-60%；Each medium sterilization
Condition be: 0.10-0.15MPa, 121 DEG C of sterilizings 30 minutes.
Step 6, by the A Bula streptomycete mycopowder 10-30 part in above-mentioned steps, field is without streptomycete mycopowder 10-30 part, soil
Earth bacillus brevis mycopowder 10-30 part, Bacillus clausii mycopowder 10-30 part, Fructus Citri tangerinae green trichoderma mycopowder 10-30 part and fulvic acid
Potassium 10-30 mixes, and packaging is composite microbic bacterial fertilizer.
Wherein, this composite microbic bacterial fertilizer for seed dressing and waters root in the method preventing and treating verticillium wilt, and the consumption of seed dressing is every thousand
Gram 50-150 gram of composite microbic bacterial fertilizer of seed is uniformly dressed seed；The using method watering root is: compound microorganism bacterium powder diluted
100-200 times, every Cotton Gossypii waters root compound microorganism bacterium powder diluent 50-100 milliliter,
This composite microbic bacterial fertilizer additionally is bottom application preventing and treating the method for verticillium wilt: uniformly applied this before ploughing compound micro-
Bio-bacterial manure 5-10kg, can preferably suppress the growth of pathogen, can play the effect of improvement soil.
The present invention has the advantage that and beneficial effect:, the A Bula streptomycete that comprises in the present invention, field without streptomycete,
Soil bacillus brevis, Bacillus clausii, five kinds of bacterium of Fructus Citri tangerinae green trichoderma are screened from the microbial strains of strain more than 450 and obtain
Energy symbiotic co-existence, can produce multiple antibiotics such as luteomycin and aburamycin；They are to suppression cotton verticillium wilt cause of disease
Bacterium--Dahlia Pinnata Cav. Verticillium has the most powerful effect, thus reaches the effect of superpower preventing and treating cotton verticillium wilt, and it is to cotton yellow
The drug effect of disease of withering is high, and average preventive effect is more than 90.0% and with strong points；, the strain of the present invention can comprehensively utilize micro-life
Antibiosis between species or in kind, competition, superparasitism, bacteriolysis, and produce disease-resistant by secondary metabolite inducing cotton
Property, strengthen its protection effect；, this 5 strain bacterial strain used in the present invention be all can be directly isolated to obtain from soil, such as soil
Earth bacillus brevis has fixed nitrogen and root mark growth-promoting functions, and other bacterium all can be numerous in crop surface, plant inside or soil
While reproductive growth, and orient plant rhizosphere and secrete some secondary metabolite, it is possible to increase plant is to the absorption of nutrient, stimulation
Plant strain growth plays the effect of fertilizer, and effect of increasing production is obvious；(4), composite microbic bacterial fertilizer of the present invention to people, animal safety, belong to
Environmentally friendly, utilize the inventive method preventing and treating cotton verticillium wilt to be not likely to produce Drug resistance, preparation method of the present invention is simple, cost
Low, use simple.
Detailed description of the invention
Embodiment 1
A kind of composite microbic bacterial fertilizer preventing and treating cotton verticillium wilt, it is characterised in that the proportioning raw materials of this microbial-bacterial fertilizer is: Ah
Bradley streptomycete mycopowder 20 parts, field is without streptomycete mycopowder 20 parts, soil bacillus brevis mycopowder 20 parts, Bacillus clausii bacterium
20 parts of powder, Fructus Citri tangerinae green trichoderma mycopowder 20 parts, potassium fulvate 20 parts；Its preparation method, comprises the following steps:
Step one, the preparation of A Bula streptomycete mycopowder
Take out A Bula streptomyces species preservation pipe, draw flat board with Gause I solid medium and recover, cultivate 7 for 30 DEG C
My god.Under flat board, the streak inoculation of picking single bacterium colony is equipped with in the Fructus Solani melongenae bottle of 150 milliliters of Gause I solid mediums, is cultivating
Case is cultivated 6-8 days for 30 DEG C, treats that lawn covers with Fructus Solani melongenae bottle, produce a large amount of spore powder and i.e. can use the physiological saline solution of 500 milliliters
Eluting, regulation spore concentration is 0.1 hundred million cfu/ml, is A Bula streptomycete seed liquor；
Secondary seed spreads cultivation: the A Bula streptomycete seed liquor of above-mentioned preparation is seeded to the A Bula of sterilizing with the inoculum concentration of 1%
On streptomycete liquid two stage seed culture medium, the fermentation tank liquid amount of 500L is 350L, ventilation be liquid-gas ratio per minute be 1:
Cultivate 40 hours for 0.8,28-30 DEG C, detect its thalline content when being 82g/L, i.e. as A Bula streptomycete secondary seed solution；
Fermentation: the A Bula streptomycete secondary seed solution of above-mentioned preparation is seeded to the inoculum concentration of 20% Ah cloth's slide fastener of sterilizing
In mould bacteria solid fermentation culture medium, after strain is mixed homogeneously with A Bula streptomycete solid medium, the A Bula planted will be connected
Streptomycete solid fermentation culture medium is spread out on fermentation tank, and height of materials is 5-10cm, temperature control 28-32 DEG C, a few days ago air humidity
Remaining 60%-75%, air humidity remains 40%-50% later, ferments 4 days, and detecting its spore content is 3,800,000,000 CFU/g, low
Warm air is done, and treats that moisture is less than 10%, pulverized 100 mesh sieves, i.e. obtains perfectly round streptomycete mycopowder；
Wherein, described Gause I solid medium: potassium nitrate: 1 gram, soluble starch: 20 grams, dipotassium hydrogen phosphate: 05,
Magnesium sulfate: 05, sodium chloride: 05, ferrous sulfate: 0 01 grams, agar: 20 grams, 1000ml, PH7 2～7 4 supplied by distilled water；
Wherein, described A Bula streptomycete secondary liquid seed culture medium: rapeseed cake powder 2%, tapioca starch 2%, sodium chloride 0.2%, carbon
Acid calcium 1%, magnesium sulfate 0.1%, PH7 2～7 4；
Wherein, described A Bula streptomycete solid fermentation culture medium: thick skin of Semen Maydis 80%, rapeseed cake 2%, tapioca starch 1%, stone powder 9%,
Wheat bran 8%, manganese sulfate 0.004%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate 0.05%, solid medium water content is 40%-60%.Each training
The condition supporting base sterilizing is: 0.10-0.15MPa, 121 DEG C of sterilizings 30 minutes.
Step 2, field is without the preparation of streptomycete mycopowder
Taking-up field, without streptomyces species preservation pipe, is drawn flat board with Gause I solid medium and is recovered, and cultivates 7 days for 30 DEG C.
Under flat board, the streak inoculation of picking single bacterium colony is equipped with in the Fructus Solani melongenae bottle of 150 milliliters of Gause I solid mediums, at incubator
In 30 DEG C cultivate 6-8 days, treat that lawn covers with Fructus Solani melongenae bottle, produce a large amount of spore powder and i.e. can use the physiological saline solution of 500 milliliters to wash
De-, regulation spore concentration is 0.1 hundred million cfu/ml, is field without streptomycete seed liquor；
Secondary seed spreads cultivation: the field of above-mentioned preparation is seeded to the field of sterilizing without strepto-without streptomycete seed liquor with the inoculum concentration of 1%
In bacteria liquid secondary seed medium, the fermentation tank liquid amount of 500L is 350L, ventilation be liquid-gas ratio per minute be 1:0.8,
Cultivate 38 hours for 28-30 DEG C, detect its thalline content when being 65g/L, i.e. as field without streptomycete secondary seed solution；
Fermentation: the field of above-mentioned preparation is seeded to the field of sterilizing without streptomycete without streptomycete secondary seed solution with the inoculum concentration of 20%
In solid fermentation culture medium, after strain is mixed homogeneously without streptomycete solid medium with field, solid without streptomycete by connecting the field planted
Body fermentation medium is spread out on fermentation tank, and height of materials is 5-10cm, and temperature control 28-32 DEG C, a few days ago air humidity remains
60%-75%, air humidity remains 40%-50% later, ferments 3-5 days, and detecting its spore content is 5,400,000,000 CFU/g, low warm air
Dry, treat that moisture is less than 10%, pulverized 100 mesh sieves, i.e. obtain field without streptomycete mycopowder；
Wherein, described Gause I solid medium: potassium nitrate: 1 gram, soluble starch: 20 grams, dipotassium hydrogen phosphate: 05,
Magnesium sulfate: 05, sodium chloride: 05, ferrous sulfate: 0 01 grams, agar: 20 grams, 1000ml, PH7 2～7 4 supplied by distilled water；
Wherein, described field is without streptomycete secondary liquid seed culture medium: cottonseed meal powder 2%, Semen Maydis powder 2%, sodium chloride 0.2%, carbonic acid
Calcium 1%, magnesium sulfate 0.1%, dipotassium hydrogen phosphate 0.1%, PH7 2～7 4；
Wherein, described field is without streptomycete solid fermentation culture medium: bagasse 80%, rapeseed cake 2%, tapioca starch 1%, stone powder 9%, slightly beautiful
Silverskin 8%, manganese sulfate 0.004%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate 0.05%, solid medium water content is 40%-60%.Each training
The condition supporting base sterilizing is: 0.10-0.15MPa, 121 DEG C of sterilizings 30 minutes.
Step 3, the preparation of soil bacillus brevis mycopowder
Taking out soil bacillus brevis preservation pipe, draw flat board respectively with nitrogen-free solid medium and recover, 30 DEG C of cultivations 72 are little
Time.Under flat board, the single colony inoculation of picking is to equipped with nitrogen-free solid medium, cultivates 72 hours in 30 DEG C of incubators, uses
3000ml sterilized water, by the lawn eluting in three Fructus Solani melongenae bottles, is seeded to equipped with 300L soil bacillus brevis seed culture medium
In 500L fermentation tank, opening stirring 120r/min, within first 8 hours, ventilation is 100L/min, and after 8-24 hour, ventilation is 200L/
Min, after 24 hours, ventilation is 300L/min, cultivates 36 hours for 30 DEG C, and detection total bacteria count content is 2,200,000,000 cfu/ml,
As soil bacillus brevis seed liquor；
Fermentation: the soil bacillus brevis seed liquor of above-mentioned preparation is seeded to soil bacillus brevis solid with the ratio of 15%
In culture medium, mixing, in tray top fermentation, windrow thickness is 5-10cm, controls fermentation temperature and is 30-40 DEG C, ferments 44 hours,
Detecting its spore content is 5,800,000,000 cfu/g, dries, and treats that moisture is less than 10%, pulverizes 100 mesh sieves, i.e. obtains the short bud of soil
Spore bacillus mycopowder；
Wherein, described nitrogen-free solid medium: sucrose 10g, dipotassium hydrogen phosphate 0.2g, magnesium sulfate 0.2g, sodium chloride 0.2g, sulphuric acid
Calcium 0.1g, calcium carbonate 5g, agar 18g, distilled water 1000mL, pH7.2；
Wherein, described soil bacillus brevis seed culture medium: molasses 20g/L, corn starch 10 g/L, cottonseed meal powder 40 g/L,
Magnesium sulfate 0.4 g/L, manganese sulfate 0.6g/L, potassium dihydrogen phosphate 0.6 g/L, calcium carbonate 10 g/L, pH7.0.Each medium sterilization
Condition be: 0.10-0.15MPa, 121 DEG C of sterilizings 30 minutes；
Soil bacillus brevis solid medium: bagasse 57%, wheat bran 35%, cottonseed meal 5%, stone powder 1%, thick skin of Semen Maydis 2%, carbamide
0.4%, dipotassium hydrogen phosphate 0.4%, magnesium sulfate 0.05%, moisture content in medium 50%-60%.The condition of each medium sterilization is:
0.10-0.15MPa, 121 DEG C of sterilizings 30 minutes
Step 4, the preparation of Bacillus clausii mycopowder
Take out Bacillus clausii preservation pipe, draw flat board respectively with nutrient solid medium and recover, 30 DEG C of cultivations
48 hours.Under flat board, the single colony inoculation of picking is to equipped with nutrient solid medium, cultivates 48 in 30 DEG C of incubators
Hour, with 3000ml sterilized water by the lawn eluting in three Fructus Solani melongenae bottles, it is seeded to equipped with 300L liquid seed culture medium
In 500L fermentation tank, opening stirring 120r/min, within first 10 hours, ventilation is 200L/min, and after 10 hours, ventilation is 320L/min
, cultivate 20 hours for 30 DEG C, detecting total bacterial content is 2,500,000,000 cfu/ml, can be used as Bacillus clausii seed liquor；
Fermentation: the Bacillus clausii seed liquor of above-mentioned preparation is added to Ke Lao with the inoculum concentration of the 10%-20% of total inoculum concentration
In family name's bacillus cereus solid fermentation culture medium, mixing, in tray top fermentation, windrow thickness is 5-10cm, controls fermentation temperature and is
30-40 DEG C, fermenting 42 hours, detection spore content is 8,400,000,000 cfu/g, dries, treats that moisture is less than 10%, pulverized 100 mesh
Sieve, i.e. obtains Bacillus clausii mycopowder；
Wherein, described nutrient broth solid medium: peptone 10.0 g/L, Carnis Bovis seu Bubali cream 3.0 g/L, sodium chloride 5.0 g/L,
Agar 20 g/L, pH 7.2 ± 0.2；
Wherein, described liquid seed culture medium: molasses 35 g/L, bean cake powder 20 g/L, magnesium sulfate 0.6g/L, manganese sulfate 0.5
G/L, potassium dihydrogen phosphate 0.3 g/L, calcium carbonate 5.0 g/L, pH7.0；
Wherein, Bacillus clausii solid fermentation culture medium: wheat bran 50%, thick skin of Semen Maydis 42%, cottonseed meal 5%, stone powder 1%, medicated beer
Grain 2%, carbamide 0.4%, dipotassium hydrogen phosphate 0.4%, magnesium sulfate 0.05%, moisture content in medium 50%-60%.The bar of each medium sterilization
Part is: 0.10-0.15MPa, 121 DEG C of sterilizings 30 minutes.
Step 5, the preparation of Fructus Citri tangerinae green trichoderma mycopowder
A, the preparation of Fructus Citri tangerinae green trichoderma seed liquor: take out Fructus Citri tangerinae green trichoderma culture presevation pipe, draw flat board with PDA solid medium and carry out multiple
Soviet Union, cultivates 6 days for 30 DEG C.Under flat board, the streak inoculation of picking single bacterium colony is equipped with the Fructus Solani melongenae bottle of 150 milliliters of PDA solid mediums
In, cultivate 4-6 days for 30 DEG C in incubator, treat that lawn covers with Fructus Solani melongenae bottle, produce a large amount of spore and i.e. can use the aseptic life of 500 milliliters
Reason salt water, regulation spore concentration is 0.1 hundred million cfu/ml, is Fructus Citri tangerinae green trichoderma seed liquor；
Fermentation: the Fructus Citri tangerinae green trichoderma seed liquor of above-mentioned preparation is seeded to the inoculum concentration of 2% the Fructus Citri tangerinae green trichoderma solid fermentation training of sterilizing
Support on base, after strain is mixed homogeneously with Fructus Citri tangerinae green trichoderma solid medium, the Fructus Citri tangerinae green trichoderma solid fermentation culture medium stand planted will be connected
On fermentation tank, height of materials is 5-10cm, temperature control 28-32 DEG C, and humidity remains 60%-75%, ferments 5 days, detects its total spore
Sub-content is 3,400,000,000 CFU/g, and low-temperature air-drying treats that moisture is less than 10%, pulverizes 100 mesh sieves, i.e. obtains Fructus Citri tangerinae green trichoderma bacterium
Powder；
Wherein, described PDA solid medium: Rhizoma Solani tuber osi 200g, sucrose 20g, water 1000mL, agar 20g；
Wherein, described Fructus Citri tangerinae green trichoderma solid fermentation culture medium: bagasse 85%, the sesame residues dregs of rice 2%, corn cob 5%, stone powder 4%, slightly
Skin of Semen Maydis 2%, carbamide 0.4%, dipotassium hydrogen phosphate 0.4%, magnesium sulfate 0.05%, moisture content in medium 50%-60%；Each medium sterilization
Condition be: 0.10-0.15MPa, 121 DEG C of sterilizings 30 minutes.
Step 6, by the A Bula streptomycete mycopowder 20 parts in above-mentioned steps, field is without streptomycete mycopowder 20 parts, the short bud of soil
Spore bacillus mycopowder 20 parts, Bacillus clausii mycopowder 20 parts, Fructus Citri tangerinae green trichoderma mycopowder 20 parts mixes with potassium fulvate 20, and packaging is i.e.
For composite microbic bacterial fertilizer.
The prevention effect of cotton verticillium wilt is tested by embodiment 2 composite microbic bacterial fertilizer
(1) test processes:
(1) test group one: the composite microbic bacterial fertilizer of embodiment 1 preparation, the consumption of seed dressing is that every kilogram of seed is combined with 100 grams
Microbial-bacterial fertilizer is uniformly dressed seed；The most normally cultivate.When cotton Seedling grows to two leaf one new film true leaves, cut root inoculation cotton yellow
Wither the spore suspension (10 of pathogen-Dahlia Pinnata Cav. lecanii strain8Individual spore/milliliter)；
(2) test group two: do not dress seed, the most normally cultivate.When cotton Seedling grows to two leaf one new film true leaves, cut root and connect
The spore suspension (10 of the former bacterium of the verticillium wilt that grows cotton-Dahlia Pinnata Cav. lecanii strain8Individual spore/milliliter) after, prepared by embodiment 1
Compound microorganism bacterium powder dilute 150 times, every Cotton Gossypii waters root compound microorganism bacterium powder diluent 60 milliliters；
(3) test group three: bottom application: uniformly applied this composite microbic bacterial fertilizer 6kg/ mu that embodiment 1 produces before ploughing, no
Seed dressing, the most normally cultivates.When cotton Seedling grows to two leaf one new film true leaves, cut root inoculation Verticillium Dahliae Infecting Cotton-
The spore suspension (10 of Dahlia Pinnata Cav. lecanii strain8Individual spore/milliliter)；
Medicament matched group: 100 times of diluent seed dressings of carbendazim, when cotton Seedling grows to two leaf one new film true leaves, cuts root inoculation
The spore suspension (108 spore/milliliters) of Verticillium Dahliae Infecting Cotton-Dahlia Pinnata Cav. lecanii strain；
Blank: clear water is dressed seed, when cotton Seedling grows to two leaf one new film true leaves, cut root inoculation Verticillium Dahliae Infecting Cotton-
The spore suspension (10 of Dahlia Pinnata Cav. lecanii strain8Individual spore/milliliter)；
Continue to cultivate when fully falling ill to blank and investigate disease index, calculate prevention effect.Often group three is parallel, Mei Geping
Row 100 strain Cotton Gossypii.
Result of the test:
The results are shown in Table 1: this composite microbic bacterial fertilizer is to dress seed in the method preventing and treating verticillium wilt, water root, or directly bottom application is improved the soil, this
Three kinds apply method, have an obvious effect relative to chemical agent, and the preventive effect of blank group relatively reached 85% with
On, effect highly significant；Comparatively speaking, carrying out bottom application better, preventive effect can reach more than 90%, and can reduce the water yield
Applying, reduce artificial, therefore recommending is bottom application；Also it can clearly be seen that use the composite microbial bacteria of the present invention from volume increase
Fertile effect is significantly higher than the prevention effect using chemical agent.
Table 1 composite microbic bacterial fertilizer is to cotton verticillium wilt efficiency test result
| Process | Disease index | Preventive effect (%) | Average product (kg) | Rate of growth (%) |
| Test group one | 12.3 | 85.59 | 8.784 | 42.04 |
| Test group two | 9.1 | 89.34 | 9.023 | 45.91 |
| Test group three | 8.2 | 90.39 | 9.488 | 53.43 |
| Chemical agent | 45.6 | 46.57 | 7.262 | 17.43 |
| Blank | 85.34 | 6.184 |
Claims (5)
1. the composite microbic bacterial fertilizer preventing and treating cotton verticillium wilt, it is characterised in that the proportioning raw materials of this microbial-bacterial fertilizer is:
A Bula streptomycete mycopowder 10-30 part, field is without streptomycete mycopowder 10-30 part, soil bacillus brevis mycopowder 10-30 part, Ke Lao
Family name's bacillus cereus mycopowder 10-30 part, Fructus Citri tangerinae green trichoderma mycopowder 10-30 part, potassium fulvate 10-30 part.
A kind of composite microbic bacterial fertilizer preventing and treating cotton verticillium wilt the most according to claim 1, it is characterised in that it is prepared
Method, comprises the following steps:
Step one, the preparation of A Bula streptomycete mycopowder
Take out A Bula streptomyces species preservation pipe, draw flat board with Gause I solid medium and recover, cultivate 7 for 30 DEG C
My god, under flat board, the streak inoculation of picking single bacterium colony is equipped with in the Fructus Solani melongenae bottle of 150 milliliters of Gause I solid mediums, is cultivating
Case is cultivated 6-8 days for 30 DEG C, treats that lawn covers with Fructus Solani melongenae bottle, produce a large amount of spore powder and i.e. can use the physiological saline solution of 500 milliliters
Eluting, regulation spore concentration is 0.1 hundred million cfu/ml, is A Bula streptomycete seed liquor；
Secondary seed spreads cultivation: the A Bula streptomycete seed liquor of above-mentioned preparation is seeded to the A Bula of sterilizing with the inoculum concentration of 1%
On streptomycete liquid two stage seed culture medium, the fermentation tank liquid amount of 500L is 350L, ventilation be liquid-gas ratio per minute be 1:
Cultivate 36-48 hour, when thalline content reaches 80g/L, can be used as A Bula streptomycete secondary seed for 0.8,28-30 DEG C
Liquid；
Fermentation: the A Bula streptomycete secondary seed solution of above-mentioned preparation is seeded to the inoculum concentration of 20% Ah cloth's slide fastener of sterilizing
In mould bacteria solid fermentation culture medium, after strain is mixed homogeneously with A Bula streptomycete solid medium, the A Bula planted will be connected
Streptomycete solid fermentation culture medium is spread out on fermentation tank, and height of materials is 5-10cm, temperature control 28-32 DEG C, a few days ago air humidity
Remaining 60%-75%, air humidity remains 40%-50% later, ferments 3-5 days, detects its spore content and is not less than 3,000,000,000
CFU/g, gets final product low-temperature air-drying, treats that moisture is less than 10%, pulverizes 100 mesh sieves, i.e. obtains A Bula streptomycete mycopowder；
Wherein, described Gause I solid medium: potassium nitrate: 1 gram, soluble starch: 20 grams, dipotassium hydrogen phosphate: 05
Gram, magnesium sulfate: 05 grams, sodium chloride: 05 grams, ferrous sulfate: 0 01 grams, agar: 20 grams, 1000ml, PH7 2 supplied by distilled water
～74；
Wherein, described A Bula streptomycete secondary liquid seed culture medium: rapeseed cake powder 2%, tapioca starch 2%, sodium chloride 0.2%, carbon
Acid calcium 1%, magnesium sulfate 0.1%, PH7 2～7 4；
Wherein, described A Bula streptomycete solid fermentation culture medium: thick skin of Semen Maydis 80%, rapeseed cake 2%, tapioca starch 1%, stone powder 9%,
Wheat bran 8%, manganese sulfate 0.004%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate 0.05%, solid medium water content is 40%-60%；Each training
The condition supporting base sterilizing is: 0.10-0.15MPa, 121 DEG C of sterilizings 30 minutes；
Step 2, field are without the preparation of streptomycete mycopowder
Taking-up field, without streptomyces species preservation pipe, is drawn flat board with Gause I solid medium and is recovered, and cultivates 7 days for 30 DEG C,
Under flat board, the streak inoculation of picking single bacterium colony is equipped with in the Fructus Solani melongenae bottle of 150 milliliters of Gause I solid mediums, at incubator
In 30 DEG C cultivate 6-8 days, treat that lawn covers with Fructus Solani melongenae bottle, produce a large amount of spore powder and i.e. can use the physiological saline solution of 500 milliliters to wash
De-, regulation spore concentration is 0.1 hundred million cfu/ml, is field without streptomycete seed liquor；
Secondary seed spreads cultivation: the field of above-mentioned preparation is seeded to the field of sterilizing without strepto-without streptomycete seed liquor with the inoculum concentration of 1%
In bacteria liquid secondary seed medium, the fermentation tank liquid amount of 500L is 350L, ventilation be liquid-gas ratio per minute be 1:0.8,
Cultivate 36-48 hour, when thalline content reaches 60g/L, can be used as field without streptomycete secondary seed solution for 28-30 DEG C；
Fermentation: the field of above-mentioned preparation is seeded to the field of sterilizing without streptomycete without streptomycete secondary seed solution with the inoculum concentration of 20%
In solid fermentation culture medium, after strain is mixed homogeneously without streptomycete solid medium with field, solid without streptomycete by connecting the field planted
Body fermentation medium is spread out on fermentation tank, and height of materials is 5-10cm, and temperature control 28-32 DEG C, a few days ago air humidity remains
60%-75%, air humidity remains 40%-50% later, ferments 3-5 days, detects its spore content and is not less than 5,000,000,000 CFU/g, i.e.
Can low-temperature air-drying, treat that moisture is less than 10%, pulverized 100 mesh sieves, i.e. obtain field without streptomycete mycopowder；
Wherein, described Gause I solid medium is identical with the Gause I solid medium in step one；
Wherein, described field is without streptomycete secondary liquid seed culture medium: cottonseed meal powder 2%, Semen Maydis powder 2%, sodium chloride 0.2%, carbonic acid
Calcium 1%, magnesium sulfate 0.1%, dipotassium hydrogen phosphate 0.1%, PH7 2～7 4；
Wherein, described field is without streptomycete solid fermentation culture medium: bagasse 80%, rapeseed cake 2%, tapioca starch 1%, stone powder 9%, slightly beautiful
Silverskin 8%, manganese sulfate 0.004%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate 0.05%, solid medium water content is 40%-60%；Each training
The condition supporting base sterilizing is: 0.10-0.15MPa, 121 DEG C of sterilizings 30 minutes；
Step 3, the preparation of soil bacillus brevis mycopowder
Taking out soil bacillus brevis preservation pipe, draw flat board respectively with nitrogen-free solid medium and recover, 30 DEG C of cultivations 72 are little
Time, under flat board, the single colony inoculation of picking is to equipped with nitrogen-free solid medium, cultivates 72 hours in 30 DEG C of incubators, uses
3000ml sterilized water, by the lawn eluting in three Fructus Solani melongenae bottles, is seeded to equipped with 300L soil bacillus brevis seed culture medium
In 500L fermentation tank, opening stirring 120r/min, within first 8 hours, ventilation is 100L/min, and after 8-24 hour, ventilation is 200L/
Min, after 24 hours, ventilation is 300L/min, cultivates 32-48 hour, treats that total bacteria count content is not less than 2,000,000,000 cfu/ for 30 DEG C
Ml, can be used as soil bacillus brevis seed liquor；
Fermentation: the soil bacillus brevis seed liquor of above-mentioned preparation is seeded to soil bacillus brevis with the ratio of 10%-20%
On solid medium, mixing, in tray top fermentation, windrow thickness is 5-10cm, controls fermentation temperature and is 30-40 DEG C, and ferment 36-
48 hours, treat that spore content is not less than 5,000,000,000 cfu/g, can dry, treat that moisture is less than 10%, pulverized 100 mesh sieves, i.e.
Obtain soil bacillus brevis mycopowder；
Wherein, described nitrogen-free solid medium: sucrose 10g, dipotassium hydrogen phosphate 0.2g, magnesium sulfate 0.2g, sodium chloride 0.2g, sulphuric acid
Calcium 0.1g, calcium carbonate 5g, agar 18g, distilled water 1000mL, pH7.2；
Wherein, described soil bacillus brevis seed culture medium: molasses 20g/L, corn starch 10 g/L, cottonseed meal powder 40 g/L,
Magnesium sulfate 0.4 g/L, manganese sulfate 0.6g/L, potassium dihydrogen phosphate 0.6 g/L, calcium carbonate 10 g/L, pH7.0, each medium sterilization
Condition be: 0.10-0.15MPa, 121 DEG C of sterilizings 30 minutes；
Wherein, described soil bacillus brevis solid medium: bagasse 57%, wheat bran 35%, cottonseed meal 5%, stone powder 1%, slightly beautiful
Silverskin 2%, carbamide 0.4%, dipotassium hydrogen phosphate 0.4%, magnesium sulfate 0.05%, moisture content in medium 50%-60%；Each medium sterilization
Condition is: 0.10-0.15MPa, 121 DEG C of sterilizings 30 minutes；
Step 4, the preparation of Bacillus clausii mycopowder
Take out Bacillus clausii preservation pipe, draw flat board respectively with nutrient solid medium and recover, 30 DEG C of cultivations
48 hours, under flat board, the single colony inoculation of picking was to equipped with nutrient solid medium, cultivates 48 in 30 DEG C of incubators
Hour, with 3000ml sterilized water by the lawn eluting in three Fructus Solani melongenae bottles, it is seeded to equipped with 300L liquid seed culture medium
In 500L fermentation tank, opening stirring 120r/min, within first 10 hours, ventilation is 200L/min, and after 10 hours, ventilation is 320L/min
, cultivate 16-24 hour, treat that total bacterial content is not less than 2,000,000,000 cfu/ml, can be used as Bacillus clausii seed liquor for 30 DEG C；
Fermentation: the Bacillus clausii seed liquor of above-mentioned preparation is added to Ke Lao with the inoculum concentration of the 10%-20% of total inoculum concentration
In family name's bacillus cereus solid fermentation culture medium, mixing, in tray top fermentation, windrow thickness is 5-10cm, controls fermentation temperature and is
30-40 DEG C, ferment 36-48 hour, treat that spore content is not less than 8,000,000,000 cfu/g, can dry, treat that moisture is less than 10%, powder
Broken mistake 100 mesh sieve, i.e. obtains Bacillus clausii mycopowder；
Wherein, described nutrient broth solid medium: peptone 10.0 g/L, Carnis Bovis seu Bubali cream 3.0 g/L, sodium chloride 5.0 g/L,
Agar 20 g/L, pH 7.2 ± 0.2；
Wherein, described liquid seed culture medium: molasses 35 g/L, bean cake powder 20 g/L, magnesium sulfate 0.6g/L, manganese sulfate 0.5
G/L, potassium dihydrogen phosphate 0.3 g/L, calcium carbonate 5.0 g/L, pH7.0；
Wherein, described Bacillus clausii solid fermentation culture medium: wheat bran 50%, thick skin of Semen Maydis 42%, cottonseed meal 5%, stone powder
1%, distiller's grains of beer 2%, carbamide 0.4%, dipotassium hydrogen phosphate 0.4%, magnesium sulfate 0.05%, moisture content in medium 50%-60%；Each culture medium
The condition of sterilizing is: 0.10-0.15MPa, 121 DEG C of sterilizings 30 minutes；
Step 5, the preparation of Fructus Citri tangerinae green trichoderma mycopowder
The preparation of Fructus Citri tangerinae green trichoderma seed liquor: take out Fructus Citri tangerinae green trichoderma culture presevation pipe, draws flat board with PDA solid medium and carries out multiple
Soviet Union, cultivates 6 days for 30 DEG C, and under flat board, the streak inoculation of picking single bacterium colony is equipped with the Fructus Solani melongenae bottle of 150 milliliters of PDA solid mediums
In, cultivate 4-6 days for 30 DEG C in incubator, treat that lawn covers with Fructus Solani melongenae bottle, produce a large amount of spore and i.e. can use the aseptic life of 500 milliliters
Reason salt water, regulation spore concentration is 0.1 hundred million cfu/ml, is Fructus Citri tangerinae green trichoderma seed liquor；
Fermentation: the Fructus Citri tangerinae green trichoderma seed liquor of above-mentioned preparation is seeded to the inoculum concentration of 2% the Fructus Citri tangerinae green trichoderma solid fermentation training of sterilizing
Support on base, after strain is mixed homogeneously with Fructus Citri tangerinae green trichoderma solid medium, the Fructus Citri tangerinae green trichoderma solid fermentation culture medium stand planted will be connected
On fermentation tank, height of materials is 5-10cm, temperature control 28-32 DEG C, and humidity remains 60%-75%, ferments 4-6 days, detects it total
Spore content is not less than 3,000,000,000 CFU/g, gets final product low-temperature air-drying, treats that moisture is less than 10%, pulverized 100 mesh sieves, and i.e. obtained Fructus Citri tangerinae
Green trichoderma mycopowder；
Wherein, described PDA solid medium: Rhizoma Solani tuber osi 200g, sucrose 20g, water 1000mL, agar 20g；
Wherein, described Fructus Citri tangerinae green trichoderma solid fermentation culture medium: bagasse 85%, the sesame residues dregs of rice 2%, corn cob 5%, stone powder 4%, slightly
Skin of Semen Maydis 2%, carbamide 0.4%, dipotassium hydrogen phosphate 0.4%, magnesium sulfate 0.05%, moisture content in medium 50%-60%；Each medium sterilization
Condition be: 0.10-0.15MPa, 121 DEG C of sterilizings 30 minutes；
Step 6, by the A Bula streptomycete mycopowder 10-30 part in above-mentioned steps, field is without streptomycete mycopowder 10-30 part, and soil is short
Bacillus cereus mycopowder 10-30 part, Bacillus clausii mycopowder 10-30 part, Fructus Citri tangerinae green trichoderma mycopowder 10-30 part and potassium fulvate
10-30 part mixes, and packaging is composite microbic bacterial fertilizer.
A kind of composite microbic bacterial fertilizer preventing and treating cotton verticillium wilt the most according to claim 1, it is characterised in that this is combined
Microbial-bacterial fertilizer is to dress seed in the method preventing and treating verticillium wilt, and the consumption of seed dressing is 50-150 gram of complex microorganism of every kilogram of seed
Bacterial manure is uniformly dressed seed.
A kind of composite microbic bacterial fertilizer preventing and treating cotton verticillium wilt the most according to claim 1, it is characterised in that this is combined
Microbial-bacterial fertilizer is preventing and treating the method for verticillium wilt for watering root, and the using method watering root is: compound microorganism bacterium powder is diluted 100-
200 times, every Cotton Gossypii waters root compound microorganism bacterium powder diluent 50-100 milliliter.
A kind of composite microbic bacterial fertilizer preventing and treating cotton verticillium wilt the most according to claim 1, it is characterised in that this is combined
Microbial-bacterial fertilizer is bottom application preventing and treating the method for verticillium wilt: uniformly applied this composite microbic bacterial fertilizer 5-10kg before ploughing.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610745953.XA CN106305793A (en) | 2016-08-29 | 2016-08-29 | Compound bacterial manure used for prevention and control of cotton verticillium wilt and the preparation method thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610745953.XA CN106305793A (en) | 2016-08-29 | 2016-08-29 | Compound bacterial manure used for prevention and control of cotton verticillium wilt and the preparation method thereof |
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| Publication Number | Publication Date |
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| CN106305793A true CN106305793A (en) | 2017-01-11 |
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| CN201610745953.XA Pending CN106305793A (en) | 2016-08-29 | 2016-08-29 | Compound bacterial manure used for prevention and control of cotton verticillium wilt and the preparation method thereof |
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| CN (1) | CN106305793A (en) |
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| CN109517744A (en) * | 2019-01-10 | 2019-03-26 | 海南大学 | The composite spore powder and its application of Trichoderma mixed cooperative solid fermentation preparation |
| CN110846243A (en) * | 2019-09-29 | 2020-02-28 | 石河子大学 | Biocontrol compound microbial inoculum and preparation method and application thereof |
| CN113527002A (en) * | 2021-04-29 | 2021-10-22 | 滨州市京阳生物肥业有限公司 | Preparation method of biological organic fertilizer for preventing and treating cotton verticillium wilt |
| CN113621540A (en) * | 2021-08-11 | 2021-11-09 | 沈阳农业大学 | Bacillus clausii strain and screening method and application thereof |
| CN113832071A (en) * | 2021-10-19 | 2021-12-24 | 新疆农业科学院微生物应用研究所(中国新疆—亚美尼亚生物工程研究开发中心) | Brevibacillus halotolerans strain and application thereof in preparation of biocontrol microbial inoculum |
| CN114436711A (en) * | 2022-01-14 | 2022-05-06 | 春华秋实科技集团有限公司 | Microbial organic fertilizer for preventing and treating cotton wilt and preparation method thereof |
| CN114574372A (en) * | 2022-03-28 | 2022-06-03 | 山东省林业科学研究院 | Trichoderma citrinoviride and application thereof in degrading fish protein |
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- 2016-08-29 CN CN201610745953.XA patent/CN106305793A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109517744A (en) * | 2019-01-10 | 2019-03-26 | 海南大学 | The composite spore powder and its application of Trichoderma mixed cooperative solid fermentation preparation |
| CN110846243A (en) * | 2019-09-29 | 2020-02-28 | 石河子大学 | Biocontrol compound microbial inoculum and preparation method and application thereof |
| CN113527002A (en) * | 2021-04-29 | 2021-10-22 | 滨州市京阳生物肥业有限公司 | Preparation method of biological organic fertilizer for preventing and treating cotton verticillium wilt |
| CN113621540A (en) * | 2021-08-11 | 2021-11-09 | 沈阳农业大学 | Bacillus clausii strain and screening method and application thereof |
| CN113621540B (en) * | 2021-08-11 | 2023-01-06 | 沈阳农业大学 | Bacillus clausii strain and screening method and application thereof |
| CN113832071A (en) * | 2021-10-19 | 2021-12-24 | 新疆农业科学院微生物应用研究所(中国新疆—亚美尼亚生物工程研究开发中心) | Brevibacillus halotolerans strain and application thereof in preparation of biocontrol microbial inoculum |
| CN113832071B (en) * | 2021-10-19 | 2023-03-10 | 新疆农业科学院微生物应用研究所(中国新疆—亚美尼亚生物工程研究开发中心) | Brevibacillus halotolerans strain and application thereof in preparation of biocontrol microbial inoculum |
| CN114436711A (en) * | 2022-01-14 | 2022-05-06 | 春华秋实科技集团有限公司 | Microbial organic fertilizer for preventing and treating cotton wilt and preparation method thereof |
| CN114574372A (en) * | 2022-03-28 | 2022-06-03 | 山东省林业科学研究院 | Trichoderma citrinoviride and application thereof in degrading fish protein |
| CN114574372B (en) * | 2022-03-28 | 2023-06-20 | 山东省林业科学研究院 | Trichoderma citrinoviride and application thereof in degradation of fish protein |
| CN115491322A (en) * | 2022-05-23 | 2022-12-20 | 安徽农业大学 | Extraction method and application of alkaloid |
| CN115491322B (en) * | 2022-05-23 | 2023-11-03 | 安徽农业大学 | Extraction method and application of alkaloid |
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Application publication date: 20170111 |