CN103651582A - Broad-spectrum composite microbial fungicide and preparation method thereof - Google Patents
Broad-spectrum composite microbial fungicide and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a broad-spectrum composite microbial fungicide and a preparation method thereof. Original composite conidial powder consists of the following three microorganisms: bacillusthuringiensis GSICC32807, bacillus amyloliquefaciens GSICC32826 and pythiumoligandrum GSICC61615. Two types of bacterial spore powder and a type of fungal spore powder are mixed according to the weight ratio of 1:1:1 to prepare a broad-spectrum composite microbial fungicide product. The broad-spectrum composite microbial fungicide product provided by the invention can be quickly planted around the root of a plant to obviously prevent, resist and kill microbial diseases of various crops by competing infection sites with pathogenic bacteria, implementing the hyperparasitism action, secreting auxin, promoting nutrient absorption of the plant and the like; furthermore, the road-spectrum composite microbial fungicide has the effects of promoting the growth of the crops, increasing the yield and improving the quality.
Description
Technical field
The invention belongs to microbial pesticide technical field, relate to a kind of wide spectrum complex microorganism bactericide and preparation method thereof.
Background technology
The whole world is because the economic loss that diseases and pests of agronomic crop causes is huge, chemical pesticide has played huge impetus to the development of the control of damage by disease and insect and agricultural production, but the mankind do not add the abuse of restraining to it, cause environmental pollution constantly to increase the weight of, person poultry poisoning frequently occurs, and also makes pest resistance to insecticide strengthen.Therefore, application is efficient, the environment protection biological agricultural chemicals of wide spectrum, for agriculture sustainable development, ensure human health and food security, safeguard that ecotope and bio-diversity are all significant.In the medium-term and long-term development in science and technology outline of country, " environmental protection type agricultural chemical initiative and the ecological agriculture " is listed in agricultural development major fields, " exploitation environmental protection type agricultural chemical initiative key technology " is its preferential theme, is also the cutting edge technology in modern agricultural technology research in country's 12 science and technology development plannings.
Summary of the invention
The object of this invention is to provide a kind of wide spectrum complex microorganism bactericide, to solve existing bactericide, make that environmental pollution constantly increases the weight of, person poultry poisoning frequently occurs, the problem that pest resistance to insecticide is strengthened.
Another object of the present invention is to provide the preparation method of this complex microorganism bactericide.
Technical solution of the present invention is as follows:
A wide spectrum complex microorganism bactericide, it is by the former semen multiplex powder of following 3 kinds of the composition of the microorganisms: bacillus thuringiensis (
bacillus thuringiensis) GSICC 32807, bacillus amyloliquefaciens (
bacillus amyloliquefaciens) GSICC 32826 and pythium oligandrum (
pythiumoligandrum) GSICC 61615.
This 3 strain bacterial classification Jun Cong Research for Industrial Microbial Germ preservation center, Gansu Province (GSMSC) is enough.
In order to realize better the present invention, wide spectrum complex microorganism bactericide also contains following composition, nutrients, wetting agent, dispersant, carrier material, and the weight proportion of each material is:
The nutrients of former semen multiplex powder 3%, 5%-10%, 5% wetting agent, 15% dispersant, the carrier material of 67%-72%, wherein nutrients is the mixture of trace element chelated dose of ammonium nitrate, potassium dihydrogen phosphate and humic acid potassium, wetting agent is lauryl sodium sulfate, dispersant is the mixture of calcium lignosulfonate and sodium methylene bis-naphthalene sulfonate, and carrier material is kaolin, diatomite or bentonite.The mass ratio that described ammonium nitrate, potassium dihydrogen phosphate and humic acid potassium are trace element chelated dose is 5.415:2.32:10, and the mass ratio of described calcium lignosulfonate and sodium methylene bis-naphthalene sulfonate is 1:1.
A preparation method for wide spectrum complex microorganism bactericide, is characterized in that it is comprised of the following step:
1) actication of culture
Because bacterial classification is preserved in 4 ℃ of strain libraries for a long time, therefore to bring back to life before use.Get 3 strain bacterial classifications in claim 1 and carry out inclined-plane cultivation, in room temperature, place after 1-2 days, be transferred to respectively test tube and carry out inclined-plane cultivation, at 25-28 ℃ of temperature, bacterial classification brings back to life in medium and cultivates 3 days, obtains activated spawn;
2) preparation of two kinds of bacterial spore powder
The bacillus thuringiensis having activated and two kinds of bacterial strains of bacillus amyloliquefaciens are inoculated into respectively in level liquid seed culture medium, are to cultivate on the shaking table of 180 revs/min at 28-30 ℃ of temperature, rotating speed; Then being transferred in secondary seed shaking flask, is to cultivate on the shaking table of 180 revs/min at 28-30 ℃ of temperature, rotating speed; Being transferred in bacterial fermentation tank medium, is to cultivate on the shaking table of 180 revs/min at 28-30 ℃ of temperature, rotating speed again; The bacterial classification of bacterial fermentation tank medium is transported to and is produced in tank, is to cultivate on the shaking table of 180 revs/min at 28-30 ℃ of temperature, rotating speed, and fermentation 2-3 days, obtains liquid bacterial suspension; Two kinds of bacterial suspensions, after concentrating low-temperature is dry, are prepared into conidial powder, containing living bacteria count >=10,000,000,000/gram;
3) preparation of fungal spore powder
Bacterial strain being cultivated in the pythium oligandrum inclined-plane that activated, be inoculated in level liquid seed culture medium, is to cultivate on the shaking table of 200 revs/min at 25-28 ℃ of temperature, rotating speed, fermentation 2-3 days; Then being transferred in secondary seed shaking flask, is to cultivate on the shaking table of 200 revs/min at 25-28 ℃ of temperature, rotating speed, and fermentation 2-3 days, then be transferred in bacterial fermentation tank medium is to cultivate on the shaking table of 200 revs/min at 25-28 ℃ of temperature, rotating speed, fermentation 2-3 days; The bacterial classification of bacterial fermentation tank medium is inoculated in solid fermentation medium, fermentation 3-5 days, form a large amount of spores, spore-bearing solid fermentation medium is infiltrated in sterile water, prepare spore suspension, then fungal spore suspension is concentrated, low temperature drying, makes fungal spore powder, spore count>=1 * 10
9individual/g;
4) complex microorganism bactericide product preparation
After three kinds of conidial powder that step 2 and 3 is obtained mix in the ratio of 1:1:1, it is former semen multiplex powder; According to following ratio, make complex microorganism bactericide product, former semen multiplex powder 3%, the nutrients of cooperation 5%-10%, 5% wetting agent, 15% dispersant, the carrier material of 65%-75%.
In this complex microorganism bactericide product: bacillus thuringiensis living bacteria count>=1 * 10
9individual/g, bacillus amyloliquefaciens living bacteria count>=1 * 10
9individual/g, pythium oligandrum spore count>=1 * 10
7the wide spectrum complex microorganism bactericide of individual/g.
In order to realize better the present invention, in step 2 and step 3, the inoculum concentration of bacillus thuringiensis and bacillus amyloliquefaciens and pythium oligandrum inclined-plane cultivation bacterial strain is 5%-10%.In step 2, the fermentation time of bacillus thuringiensis and two kinds of bacterial strains of bacillus amyloliquefaciens is 2 days.
In order to realize better the present invention, in step 1, bacterial classification brings back to life culture medium prescription: potato 20%, glucose 2%, beef extract 0.3%, peptone 1%, sodium chloride 0.5%, agar 1.5%, concrete making: get a certain amount of potato, remove the peel, be cut into piece and boil 30min, then use 200 order filtered through gauze, add again above-mentioned other raw materials to supply water, supply water, packing test tube after agar dissolves, 121 ℃ of sterilizing 30min.
In order to realize better the present invention, liquid seed culture medium formula in step 2: potato 20%, glucose 2%, beef extract 0.3%, peptone 1%, sodium chloride 0.5%, concrete making: get the potato of 200g peeling, be cut into piece and boil 30min, then use 200 order filtered through gauze, add again above-mentioned other raw materials to supply water, 121 ℃ of sterilizing 30min.
In order to realize better the present invention, the bacterial fermentation tank medium in step 2 and 3 forms and mass content is: molasses 3-5%, beef extract 0.3%, peptone 1%, sodium chloride 0.5%, magnesium sulfate 0.05%, potassium dihydrogen phosphate 0.05%, potassium nitrate 0.1%, adds water and supplies, and pH value is 6.8-7.4.
In order to realize better the present invention, solid fermentation culture medium prescription in step 2: soya-bean cake 5%, corn flour 10%, wheat bran 50%, powder of straw 35%, wherein the mass ratio of solid material and moisture is 3:2.
The using method of microbial inoculum of the present invention can ditch spread, spread manuer in holes, pesticide-clay mixture, soak seed, dip in root, spray, fill with root.
The mechanism of microbial inoculum of the present invention is as follows:
Bacillus thuringiensis (
bacillus thuringiensis) be called for short Bt, be that current production rate is maximum, most popular lepidopterous insect pest biocontrol agent.But the control efficiency of Bt is affected by environment larger, the shortcoming such as quick-acting is poor, easily produce resistance, ecological niche is little, affects Bt product large-scale promotion application.Bacillus amyloliquefaciens (
bacillus amyloliquefaciens) be the bacterium that a strain and bacillus subtilis affinity are very high, the diseases such as fungus-caused rice blast, banded sclerotial blight, fusarium wilt and gray mold are all had to control efficiency.But due to bacillus amyloliquefaciens, affected by temperature and humidity larger, and ecological niche is little, therefore limited to the control efficiency of crop fungal disease, restrict the production application of this bacterial strain.
Pythium oligandrum (
pythiumoligandrum) be a kind of pythium, identified that at present pythium has kind more than 200, great majority are all the pathogenic bacteria of plant, but pythium oligandrum is but the biocontrol microorganisms of a lot of plants.Because pythium oligandrum belongs to pythium, so a lot of plants are had to the parasitism of wide spectrum, after parasitism, can obviously improve the disease resistance of plant.Research shows, pythium oligandrum is to the bacterium of the plants such as tomato, potato, paddy rice, cucumber and grape and well effect of fungal disease control performance.Pythium oligandrum cell wall protein is intended hidden ground albumen (CWPs) can act on LeATL6 ubiquitin protein ligase E3 induced defense responses.Also studies have found that pythium oligandrum, when control tomato bacterialo wilt disease, has dependence to blackish green acid signal.Visible pythium oligandrum is realized Biocontrol Effect by strengthening the system of defense of plant self, therefore the product of plant and quality are had to safety, and pythium oligandrum is fungus biocontrol agent, easily cultivate, breeding is fast, ecological niche is large, is applicable to the exploitation of biocontrol agent.
The damage by disease and insect majority of crop is after harmful insect bite is bitten, for pathogen provides the invasion integrated disease that chance causes.The present invention is according to its pathogenic feature, utilize the synergitic effect of bacillus thuringiensis, bacillus amyloliquefaciens and pythium oligandrum, exploitation has the wide spectrum complex microorganism bactericide product of desinsection and control microorganism disease targetedly, make product of the present invention solve existing bactericide and make that environmental pollution constantly increases the weight of, person poultry poisoning frequently occurs, the problem that pest resistance to insecticide is strengthened.Wide spectrum complex microorganism bactericide product provided by the invention rapidly field planting arrives plant rhizosphere, by infecting site, hyperparasitism, secretion growth hormone and promote the effects such as plant nutrition absorption with pathogen competition, remarkable prevention, antagonism are also killed various crop microorganism disease; Have simultaneously and promote raw crop to grow, improve output and improve the effect of quality.
The plant beneficial microorganism that the selected microorganism fungus kind Jun Shi Ministry of Agriculture of the present invention is authorized, through the composite wide spectrum complex microorganism bactericide product of making, has the following advantages:
1, rationally, bacillus thuringiensis can prevent lepidopterous insect pest in microbe species collocation of the present invention, and minimizing insect stings " the convenient passage " providing for pathogen invasion is provided; Bacillus amyloliquefaciens secretion high-temperature resistant antibacterial material, protease and growth hormone etc., bring into play certain biological and ecological methods to prevent plant disease, pests, and erosion growth-promoting functions to disease control, but its ecological niche little, be subject to such environmental effects; And pythium oligandrum further supplements the function of bacillus amyloliquefaciens, its ecological niche is large, easily breeding, and host is extensive.Triplicity to the extermination of disease and insect pest have efficiently, the effect of wide spectrum.
2, broad-spectrum micro-organisms bactericide of the present invention is selected at nutriment and carrier material, fully takes into account bacterium and fungi to the antagonism between the demand of nutriment and microorganism, synergistic effect.Complex micro organism fungicide preparation method of the present invention is simple and feasible, and effectively spore count content is high, and the microbial survival time is long, and product is stable.
3, product of the present invention is easy to use, not only can prevent insect pest, bacterium and fungus-caused disease, and can also promote plant growth, raising crop yield and quality, is the green companion of modern organic farming.
Embodiment
First from Research for Industrial Microbial Germ preservation center, Gansu Province (GSMSC), obtaining the inclined-plane of above-mentioned 3 bacterial classifications cultivates.Because bacterial classification is preserved in 4 ℃ of strain libraries for a long time, therefore to bring back to life before use.Concrete rejuvenation method: get above bacterial classification from preservation center, place after 1-2 days in room temperature, be transferred to respectively test tube slant and cultivate, cultivate 3 days, just obtain activated spawn for 25-28 ℃.
1L bacterial classification brings back to life culture medium prescription: potato 200g, glucose 20g, beef extract 3g, peptone 10g, sodium chloride 5g, agar 15g.Concrete making: get the potato of 200g peeling, be cut into piece and boil 30min, then use 200 order filtered through gauze, then add above-mentioned other raw materials, supply water to 1L after agar dissolves, packing test tube, 121 ℃ of sterilizing 30min.
Embodiment 1
1) preparation of two kinds of bacterial spore powder
Bacterial strains are cultivated in two kinds of inclined-planes of the bacillus thuringiensis having activated and bacillus amyloliquefaciens, and the inoculum concentration according to 5% is inoculated into respectively in 500mL level liquid seed culture medium, on the shaking table that 28-30 ℃, rotating speed are 180 revs/min, cultivates, and ferments 2 days.Then be transferred in secondary seed shaking flask, on the shaking table of 180 revs/min of 28-30 ℃, rotating speed, cultivate, ferment 2 days.Be transferred to again in 50L bacterial fermentation tank medium, temperature 28-30 ℃, 180 revs/min of rotating speeds, ferment 2 days.The bacterial classification of bacterial fermentation tank medium is transported to 500L and produces in tank, temperature 28-30 ℃, 180 revs/min of rotating speeds, ferment 3 days, can obtain liquid bacterial suspension.Two kinds of bacterial suspensions, after concentrating low-temperature is dry, are prepared into conidial powder, and bacillus thuringiensis is containing 10,000,000,000/gram of living bacteria counts; Bacillus amyloliquefaciens is containing 10,800,000,000/gram of living bacteria counts.
2) preparation of fungal spore powder
Bacterial strain is cultivated in the pythium oligandrum inclined-plane that activated, and the inoculum concentration according to 5% is inoculated into respectively in 500mL level liquid seed culture medium, on the shaking table that 25-28 ℃, rotating speed are 200 revs/min, cultivates, and ferments 2 days.Then be transferred in secondary seed shaking flask, on the shaking table of 200 revs/min of 25-28 ℃, rotating speed, cultivate, ferment 2 days.Be transferred to again in 50L bacterial fermentation tank medium, temperature 25-28 ℃, 200 revs/min of rotating speeds, ferment 2 days.The bacterial classification of bacterial fermentation tank medium is inoculated in solid fermentation medium, ferments and can form a large amount of spores in 3 days.The solid fermentation medium that produces spore is infiltrated in sterile water, prepare spore suspension, then, by further concentrated, the low temperature drying of fungal spore suspension, make fungal spore powder, spore count is 1.5 * 10
9individual/g.
3) complex microorganism bactericide product preparation
After three kinds of conidial powder mix in the ratio of 1:1:1, it is former semen multiplex powder.In complex microorganism bactericide product, contain former semen multiplex powder 3%, the wetting agent of nutrients 5%, 5%, 15% dispersant, carrier material 72%.Wherein nutrients is that trace element chelated dose of mass ratio according to 5.415:2.32:10 of ammonium nitrate, potassium dihydrogen phosphate and humic acid potassium mixes; Wetting agent is lauryl sodium sulfate; Dispersant is that the quality of calcium lignosulfonate and sodium methylene bis-naphthalene sulfonate 1:1 compares mixture; Carrier material is mainly kaolin, diatomite or bentonite.According to its ratio is composite, can obtain bacillus thuringiensis living bacteria count 1.2 * 10
9individual/g, bacillus amyloliquefaciens living bacteria count 1.2 * 10
9individual/g, pythium oligandrum spore count 5.6 * 10
7the wide spectrum complex microorganism bactericide of individual/g.
Embodiment 2
1) preparation of two kinds of bacterial spore powder
Bacterial strains are cultivated in two kinds of inclined-planes of the bacillus thuringiensis having activated and bacillus amyloliquefaciens, and the inoculum concentration according to 8% is inoculated into respectively in 500mL level liquid seed culture medium, on the shaking table that 28-30 ℃, rotating speed are 180 revs/min, cultivates, and ferments 2 days.Then be transferred in secondary seed shaking flask, on the shaking table of 180 revs/min of 28-30 ℃, rotating speed, cultivate, ferment 2 days.Be transferred to again in 50L bacterial fermentation tank medium, temperature 28-30 ℃, 180 revs/min of rotating speeds, ferment 2 days.The bacterial classification of bacterial fermentation tank medium is transported to 500L and produces in tank, temperature 28-30 ℃, 180 revs/min of rotating speeds, ferment 3 days, can obtain liquid bacterial suspension.Two kinds of bacterial suspensions, after concentrating low-temperature is dry, are prepared into conidial powder, and bacillus thuringiensis is containing 11,000,000,000/gram of living bacteria counts; Bacillus amyloliquefaciens is containing 11,200,000,000/gram of living bacteria counts.
2) preparation of fungal spore powder
Bacterial strain is cultivated in the pythium oligandrum inclined-plane that activated, and the inoculum concentration according to 8% is inoculated into respectively in 500mL level liquid seed culture medium, on the shaking table that 25-28 ℃, rotating speed are 200 revs/min, cultivates, and ferments 2 days.Then be transferred in secondary seed shaking flask, on the shaking table of 200 revs/min of 25-28 ℃, rotating speed, cultivate, ferment 2 days.Be transferred to again in 50L bacterial fermentation tank medium, temperature 25-28 ℃, 200 revs/min of rotating speeds, ferment 2 days.The bacterial classification of bacterial fermentation tank medium is inoculated in solid fermentation medium, ferments and can form a large amount of spores in 3 days.The solid fermentation medium that produces spore is infiltrated in sterile water, prepare spore suspension, then, by further concentrated, the low temperature drying of fungal spore suspension, make fungal spore powder, spore count is 2.0 * 10
9individual/g.
3) complex microorganism bactericide product preparation
After three kinds of conidial powder mix in the ratio of 1:1:1, it is former semen multiplex powder.In complex microorganism bactericide product, contain former semen multiplex powder 3%, the wetting agent of nutrients 10%, 5%, 15% dispersant, carrier material 67%.Wherein nutrients is that trace element chelated dose of mass ratio according to 5.415:2.32:10 of ammonium nitrate, potassium dihydrogen phosphate and humic acid potassium mixes; Wetting agent is lauryl sodium sulfate; Dispersant is that the quality of calcium lignosulfonate and sodium methylene bis-naphthalene sulfonate 1:1 compares mixture; Carrier material is mainly kaolin, diatomite or bentonite.According to its ratio is composite, can obtain bacillus thuringiensis living bacteria count 1.8 * 10
9individual/g, bacillus amyloliquefaciens living bacteria count 1.5 * 10
9individual/g, pythium oligandrum spore count 6 * 10
7the wide spectrum complex microorganism bactericide of individual/g.
Embodiment 3
1) preparation of two kinds of bacterial spore powder
Bacterial strains are cultivated in two kinds of inclined-planes of the bacillus thuringiensis having activated and bacillus amyloliquefaciens, and the inoculum concentration according to 10% is inoculated into respectively in 500mL level liquid seed culture medium, on the shaking table that 28-30 ℃, rotating speed are 180 revs/min, cultivates, and ferments 2 days.Then be transferred in secondary seed shaking flask, on the shaking table of 180 revs/min of 28-30 ℃, rotating speed, cultivate, ferment 2 days.Be transferred to again in 50L bacterial fermentation tank medium, temperature 28-30 ℃, 180 revs/min of rotating speeds, ferment 2 days.The bacterial classification of bacterial fermentation tank medium is transported to 500L and produces in tank, temperature 28-30 ℃, 180 revs/min of rotating speeds, ferment 3 days, can obtain liquid bacterial suspension.Two kinds of bacterial suspensions, after concentrating low-temperature is dry, are prepared into conidial powder, and bacillus thuringiensis is containing 11,300,000,000/gram of living bacteria counts; Bacillus amyloliquefaciens is containing 11,500,000,000/gram of living bacteria counts.
2) preparation of fungal spore powder
Bacterial strain is cultivated in the pythium oligandrum inclined-plane that activated, and the inoculum concentration according to 10% is inoculated into respectively in 500mL level liquid seed culture medium, on the shaking table that 25-28 ℃, rotating speed are 200 revs/min, cultivates, and ferments 2 days.Then be transferred in secondary seed shaking flask, on the shaking table of 200 revs/min of 25-28 ℃, rotating speed, cultivate, ferment 2 days.Be transferred to again in 50L bacterial fermentation tank medium, temperature 25-28 ℃, 200 revs/min of rotating speeds, ferment 2 days.The bacterial classification of bacterial fermentation tank medium is inoculated in solid fermentation medium, ferments and can form a large amount of spores in 3 days.The solid fermentation medium that produces spore is infiltrated in sterile water, prepare spore suspension, then, by further concentrated, the low temperature drying of fungal spore suspension, make fungal spore powder, spore count is 2.2 * 10
9individual/g.
3) complex microorganism bactericide product preparation
After three kinds of conidial powder mix in the ratio of 1:1:1, it is former semen multiplex powder.In complex microorganism bactericide product, contain former semen multiplex powder 3%, the wetting agent of nutrients 8%, 5%, 15% dispersant, carrier material 69%.Wherein nutrients is that trace element chelated dose of mass ratio according to 5.415:2.32:10 of ammonium nitrate, potassium dihydrogen phosphate and humic acid potassium mixes; Wetting agent is lauryl sodium sulfate; Dispersant is that the quality of calcium lignosulfonate and sodium methylene bis-naphthalene sulfonate 1:1 compares mixture; Carrier material is mainly kaolin, diatomite or bentonite.According to its ratio is composite, can obtain bacillus thuringiensis living bacteria count 1.52 * 10
9individual/g, bacillus amyloliquefaciens living bacteria count 1.38 * 10
9individual/g, pythium oligandrum spore count 6 * 10
7the wide spectrum complex microorganism bactericide of individual/g.
Experimental result shows: nutraceutical adding proportion has impact to complex micro organism fungicide effective microbe quantity, and nutritive proportion is higher, and effective microbe quantity increases to some extent, shows that nutritive proportion content is high, and when nutritious, microorganism has slight propagation situation.In this scope of experiment, complex microorganism bactericide product contains bacillus thuringiensis living bacteria count>=1 * 10
9individual/g, bacillus amyloliquefaciens living bacteria count>=1 * 10
9individual/g; Pythium oligandrum spore count>=1 * 10
7individual/g.This complex microorganism bactericide product index meets bactericide product requirement.
Embodiment 4
1) preparation of two kinds of bacterial spore powder
Bacterial strains are cultivated in two kinds of inclined-planes of the bacillus thuringiensis having activated and bacillus amyloliquefaciens, and the inoculum concentration according to 8% is inoculated into respectively in 500mL level liquid seed culture medium, on the shaking table that 28-30 ℃, rotating speed are 180 revs/min, cultivates, and ferments 2 days.Then be transferred in secondary seed shaking flask, on the shaking table of 180 revs/min of 28-30 ℃, rotating speed, cultivate, ferment 2 days.Be transferred to again in 50L bacterial fermentation tank medium, temperature 28-30 ℃, 180 revs/min of rotating speeds, ferment 2 days.The bacterial classification of bacterial fermentation tank medium is transported to 500L and produces in tank, temperature 28-30 ℃, 180 revs/min of rotating speeds, ferment 1 day, can obtain liquid bacterial suspension.Two kinds of bacterial suspensions, after concentrating low-temperature is dry, are prepared into conidial powder, and bacillus thuringiensis is containing 8,000,000,000/gram of living bacteria counts; Bacillus amyloliquefaciens is containing 9,600,000,000/gram of living bacteria counts.
2) preparation of fungal spore powder
Bacterial strain is cultivated in the pythium oligandrum inclined-plane that activated, and the inoculum concentration according to 5% is inoculated into respectively in 500mL level liquid seed culture medium, on the shaking table that 25-28 ℃, rotating speed are 200 revs/min, cultivates, and ferments 3 days.Then be transferred in secondary seed shaking flask, on the shaking table of 200 revs/min of 25-28 ℃, rotating speed, cultivate, ferment 3 days.Be transferred to again in 50L bacterial fermentation tank medium, temperature 25-28 ℃, 200 revs/min of rotating speeds, ferment 3 days.The bacterial classification of bacterial fermentation tank medium is inoculated in solid fermentation medium, ferments and can form a large amount of spores in 5 days.The solid fermentation medium that produces spore is infiltrated in sterile water, prepare spore suspension, then, by further concentrated, the low temperature drying of fungal spore suspension, make fungal spore powder, spore count is 6.4 * 10
9individual/g.
3) complex microorganism bactericide product preparation
After three kinds of conidial powder mix in the ratio of 1:1:1, it is former semen multiplex powder.In complex microorganism bactericide product, contain former semen multiplex powder 3%, the wetting agent of nutrients 5%, 5%, 15% dispersant, carrier material 72%.Wherein nutrients is that trace element chelated dose of mass ratio according to 5.415:2.32:10 of ammonium nitrate, potassium dihydrogen phosphate and humic acid potassium mixes; Wetting agent is lauryl sodium sulfate; Dispersant is that the quality of calcium lignosulfonate and sodium methylene bis-naphthalene sulfonate 1:1 compares mixture; Carrier material is mainly kaolin, diatomite or bentonite.According to its ratio is composite, can obtain bacillus thuringiensis living bacteria count 1.2 * 10
9individual/g, bacillus amyloliquefaciens living bacteria count 1.2 * 10
9individual/g, pythium oligandrum spore count 5.6 * 10
7the wide spectrum complex microorganism bactericide of individual/g.
Embodiment 5
1) preparation of two kinds of bacterial spore powder
Bacterial strains are cultivated in two kinds of inclined-planes of the bacillus thuringiensis having activated and bacillus amyloliquefaciens, and the inoculum concentration according to 8% is inoculated into respectively in 500mL level liquid seed culture medium, on the shaking table that 28-30 ℃, rotating speed are 180 revs/min, cultivates, and ferments 2 days.Then be transferred in secondary seed shaking flask, on the shaking table of 180 revs/min of 28-30 ℃, rotating speed, cultivate, ferment 2 days.Be transferred to again in 50L bacterial fermentation tank medium, temperature 28-30 ℃, 180 revs/min of rotating speeds, ferment 2 days.The bacterial classification of bacterial fermentation tank medium is transported to 500L and produces in tank, temperature 28-30 ℃, 180 revs/min of rotating speeds, ferment 2 days, can obtain liquid bacterial suspension.Two kinds of bacterial suspensions, after concentrating low-temperature is dry, are prepared into conidial powder, and bacillus thuringiensis is containing 13,000,000,000/gram of living bacteria counts; Bacillus amyloliquefaciens is containing 12,400,000,000/gram of living bacteria counts.
2) preparation of fungal spore powder
Bacterial strain is cultivated in the pythium oligandrum inclined-plane that activated, and the inoculum concentration according to 8% is inoculated into respectively in 500mL level liquid seed culture medium, on the shaking table that 25-28 ℃, rotating speed are 200 revs/min, cultivates, and ferments 3 days.Then be transferred in secondary seed shaking flask, on the shaking table of 200 revs/min of 25-28 ℃, rotating speed, cultivate, ferment 3 days.Be transferred to again in 50L bacterial fermentation tank medium, temperature 25-28 ℃, 200 revs/min of rotating speeds, ferment 3 days.The bacterial classification of bacterial fermentation tank medium is inoculated in solid fermentation medium, ferments and can form a large amount of spores in 5 days.The solid fermentation medium that produces spore is infiltrated in sterile water, prepare spore suspension, then, by further concentrated, the low temperature drying of fungal spore suspension, make fungal spore powder, spore count is 6.8 * 10
9individual/g.
3) complex microorganism bactericide product preparation
After three kinds of conidial powder mix in the ratio of 1:1:1, it is former semen multiplex powder.In complex microorganism bactericide product, contain former semen multiplex powder 3%, the wetting agent of nutrients 10%, 5%, 15% dispersant, carrier material 67%.Wherein nutrients is that trace element chelated dose of mass ratio according to 5.415:2.32:10 of ammonium nitrate, potassium dihydrogen phosphate and humic acid potassium mixes; Wetting agent is lauryl sodium sulfate; Dispersant is that the quality of calcium lignosulfonate and sodium methylene bis-naphthalene sulfonate 1:1 compares mixture; Carrier material is mainly kaolin, diatomite or bentonite.According to its ratio is composite, can obtain bacillus thuringiensis living bacteria count 1.8 * 10
9individual/g, bacillus amyloliquefaciens living bacteria count 1.5 * 10
9individual/g, pythium oligandrum spore count 6 * 10
7the wide spectrum complex microorganism bactericide of individual/g.
Embodiment 6
1) preparation of two kinds of bacterial spore powder
Bacterial strains are cultivated in two kinds of inclined-planes of the bacillus thuringiensis having activated and bacillus amyloliquefaciens, and the inoculum concentration according to 8% is inoculated into respectively in 500mL level liquid seed culture medium, on the shaking table that 28-30 ℃, rotating speed are 180 revs/min, cultivates, and ferments 2 days.Then be transferred in secondary seed shaking flask, on the shaking table of 180 revs/min of 28-30 ℃, rotating speed, cultivate, ferment 2 days.Be transferred to again in 50L bacterial fermentation tank medium, temperature 28-30 ℃, 180 revs/min of rotating speeds, ferment 2 days.The bacterial classification of bacterial fermentation tank medium is transported to 500L and produces in tank, temperature 28-30 ℃, 180 revs/min of rotating speeds, ferment 3 days, can obtain liquid bacterial suspension.Two kinds of bacterial suspensions, after concentrating low-temperature is dry, are prepared into conidial powder, and bacillus thuringiensis is containing 10,800,000,000/gram of living bacteria counts; Bacillus amyloliquefaciens is containing 11,200,000,000/gram of living bacteria counts.
Experimental result shows: inoculum concentration is little on the impact of two kinds of effective bacterium numbers of conidial powder, the ferment tank time is larger on the effective bacterium number impact of conidial powder, during second day, living bacteria count is maximum, along with the prolongation of fermentation time, because bacterium self-dissolving makes living bacteria count, declines a little.Therefore, the best preparation technology of two kinds of bacterial spore powder is inoculum concentration 5%-10%, equal 2 days the bests of fermentation time.
2) preparation of fungal spore powder
Bacterial strain is cultivated in the pythium oligandrum inclined-plane that activated, and the inoculum concentration according to 10% is inoculated into respectively in 500mL level liquid seed culture medium, on the shaking table that 25-28 ℃, rotating speed are 200 revs/min, cultivates, and ferments 3 days.Then be transferred in secondary seed shaking flask, on the shaking table of 200 revs/min of 25-28 ℃, rotating speed, cultivate, ferment 3 days.Be transferred to again in 50L bacterial fermentation tank medium, temperature 25-28 ℃, 200 revs/min of rotating speeds, ferment 3 days.The bacterial classification of bacterial fermentation tank medium is inoculated in solid fermentation medium, ferments and can form a large amount of spores in 5 days.The solid fermentation medium that produces spore is infiltrated in sterile water, prepare spore suspension, then, by further concentrated, the low temperature drying of fungal spore suspension, make fungal spore powder, spore count is 6.5 * 10
9individual/g.
Experimental result shows: inoculum concentration is little on the impact of pythium oligandrum spore count, and solid fermentation incubation time is larger on spore count impact, and the 5th day spore count be apparently higher than the 3rd day, shows that mycelia is aging, nutrients reduces and is conducive to sporulation in medium.Therefore, the best preparation technology of pythium oligandrum conidial powder is inoculum concentration 5%-10%, 5 days the bests of solid fermentation incubation time.
3) complex microorganism bactericide product preparation
After three kinds of conidial powder mix in the ratio of 1:1:1, it is former semen multiplex powder.In complex microorganism bactericide product, contain former semen multiplex powder 3%, the wetting agent of nutrients 8%, 5%, 15% dispersant, carrier material 69%.Wherein nutrients is that trace element chelated dose of mass ratio according to 5.415:2.32:10 of ammonium nitrate, potassium dihydrogen phosphate and humic acid potassium mixes; Wetting agent is lauryl sodium sulfate; Dispersant is that the quality of calcium lignosulfonate and sodium methylene bis-naphthalene sulfonate 1:1 compares mixture; Carrier material is mainly kaolin, diatomite or bentonite.According to its ratio is composite, can obtain bacillus thuringiensis living bacteria count 1.52 * 10
9individual/g, bacillus amyloliquefaciens living bacteria count 1.38 * 10
9individual/g, pythium oligandrum spore count 6 * 10
7the wide spectrum complex microorganism bactericide of individual/g.
Experimental result shows: nutraceutical adding proportion has impact to complex micro organism fungicide effective microbe quantity, and nutritive proportion is higher, and effective microbe quantity increases to some extent, shows that nutritive proportion content is high, and when nutritious, microorganism has slight propagation situation.In this scope of experiment, complex microorganism bactericide product contains bacillus thuringiensis living bacteria count>=1 * 10
9individual/g, bacillus amyloliquefaciens living bacteria count>=1 * 10
9individual/g; Pythium oligandrum spore count>=1 * 10
7individual/g.This complex microorganism bactericide product index meets bactericide product requirement.
practical example 1: the effect of complex microorganism bactericide to wheat diseases control and volume increase
Potted plant experiment: from the separated pathogen strain bacterium of wheat root, through be accredited as strong male rotten mould (
pythiumarrhenomanes).Contrast (CK): aseptic soil matrix; Experimental group 1 (P.A): the sterilizing soil matrix matter of the rotten mould pathogenic bacteria of strong hero of interpolation 5 ‰; Experimental group 2 (P.O): the sterilizing soil matrix matter of the complex micro organism fungicide product of interpolation 5 ‰; Experimental group 3 (P.O+P.A): the sterilizing soil matrix matter of jointly adding the complex micro organism fungicide product of 2.5 ‰ rotten mould pathogenic bacteria of strong hero and 2.5 ‰.9 repetitions are processed in each experiment, water respectively after 1L water, plant spring wheat L8275, hot-house culture.Result is as table 1: the diseases prevention of complex micro organism fungicide product and growth-promoting effect.
Experimental result shows, complex micro organism fungicide product energy wheat growth-promoting regulates neck pH value, promotes to tiller, and the rotten mould disease control rate of strong hero is reached to 60%.
Field trial: after composite bacteria agent capable product mixes with chemical fertilizer, execute field with the form of base fertilizer, 50 grams/acre of microbial inoculum usage amounts.Winter wheat rust control efficiency is reached to 70%, reduce 2/3 of chemical pesticide consumption, finally to wheat per mu yield volume increase 90-100 kilogram.
practical example 2: the effect of complex microorganism bactericide to pepper diseases control and volume increase
During pepper seedling raising, be evenly sprinkling upon field, nursery after microbial bacterial agent product is mixed to soil, applied amount is that 2 grams of complex micro organism fungicide products are added in every square metre of nursery, then ploughs, whole ground, sowing.Complex micro organism fungicide has the effect that improves emergence rate, promotes to grow, shorten seedling raise period, compares with not adding contrasting of this product, can carry and emerging the last week.Be transplanted to behind field, except promoting the effect of growth, can also be more solid than Zao 10 days of the contrast of not adding microbial inoculum product, and prolongation growth cycle 10-15 days.The effect in early days with the fusarium wilt of preventing and treating and powdery mildew, remarkable to the control efficiency of pepper fruit brown spot and rot in the later stage, can reduce 4/5 of chemical pesticide consumption.Complex microorganism bactericide is not only remarkable to the disease-controlling effect of capsicum whole breeding time, and can also increase production and improve quality, thousand grams/acre of units increased in production 150-180.
practical example 3: the effect of complex microorganism bactericide to Radix Angelicae Sinensis disease control and volume increase
After complex microorganism bactericide is mixed according to the ratio of 1:100 with soil, in the mode of pesticide-clay mixture, add (consistent with 30% chlopyrifos wetting powder insecticide-applying way) on the seedling of cave to.Result shows, after complex microorganism bactericide is processed, the Radix Angelicae Sinensis fiber crops stomatosis incidence of disease is 7%, and the incidence of disease of chemical pesticide is 16%.Complex microorganism bactericide is better than chemical pesticide chlopyrifos to Radix Angelicae Sinensis fiber crops stomatosis disease-controlling effect, and has increased production 8%.After complex micro organism fungicide is processed, not only can increase output, and significantly improve first-class and return and second-class quantum of output of returning.
The effect experimental result of above wide spectrum complex microorganism bactericide of the present invention shows:
1, add after complex microorganism bactericide of the present invention, can obviously improve crop disease-resistant ability.Not only the strong male rotten mould root rot causing is had to good control efficiency, and remarkable to the brown spot of numb stomatosis, powdery mildew and pepper fruit and preventing and treating rot disease effect.Show that this product has broad spectrum activity to disease control.
2, add after complex microorganism bactericide of the present invention, the effect of increasing income is remarkable.Show that the microorganism that this product is selected is the probio of crop, to the growth of crop, yield and quality performance beneficial effect.
3, this complex microorganism bactericide is applied widely, can be used for the aspects such as crops, melon and fruit, vegetables and Special Traditional Chinese Medicine material.
4, this microbial inoculum product is pressed powder, and convenient transportation is used simple to operately, easily promote the wider application.
Claims (10)
1. a wide spectrum complex microorganism bactericide, is characterized in that: it is by the former semen multiplex powder of following 3 kinds of the composition of the microorganisms: bacillus thuringiensis (
bacillus thuringiensis) GSICC 32807, bacillus amyloliquefaciens (
bacillus amyloliquefaciens) GSICC 32826 and pythium oligandrum (
pythiumoligandrum) GSICC 61615.
2. wide spectrum complex microorganism bactericide according to claim 1, is characterized in that: it also contains following composition, nutrients, wetting agent, dispersant, carrier material, and the weight proportion of each material is:
The nutrients of former semen multiplex powder 3%, 5%-10%, 5% wetting agent, 15% dispersant, the carrier material of 67%-72%, wherein nutrients is the mixture of trace element chelated dose of ammonium nitrate, potassium dihydrogen phosphate and humic acid potassium, wetting agent is lauryl sodium sulfate, dispersant is the mixture of calcium lignosulfonate and sodium methylene bis-naphthalene sulfonate, and carrier material is kaolin, diatomite or bentonite.
3. wide spectrum complex microorganism bactericide according to claim 2, it is characterized in that: the mass ratio that described ammonium nitrate, potassium dihydrogen phosphate and humic acid potassium are trace element chelated dose is 5.415:2.32:10, the mass ratio of described calcium lignosulfonate and sodium methylene bis-naphthalene sulfonate is 1:1.
4. the preparation method of wide spectrum complex microorganism bactericide described in claim 1, is characterized in that it is comprised of the following step:
1) actication of culture
Get 3 strain bacterial classifications in claim 1 and carry out inclined-plane cultivation, in room temperature, place after 1-2 days, be transferred to respectively test tube and carry out inclined-plane cultivation, at 25-28 ℃ of temperature, bacterial classification brings back to life in medium and cultivates 3 days, obtains activated spawn;
2) preparation of two kinds of bacterial spore powder
The bacillus thuringiensis having activated and two kinds of bacterial strains of bacillus amyloliquefaciens are inoculated into respectively in level liquid seed culture medium, are to cultivate on the shaking table of 180 revs/min at 28-30 ℃ of temperature, rotating speed; Then being transferred in secondary seed shaking flask, is to cultivate on the shaking table of 180 revs/min at 28-30 ℃ of temperature, rotating speed; Being transferred in bacterial fermentation tank medium, is to cultivate on the shaking table of 180 revs/min at 28-30 ℃ of temperature, rotating speed again; The bacterial classification of bacterial fermentation tank medium is transported to and is produced in tank, is to cultivate on the shaking table of 180 revs/min at 28-30 ℃ of temperature, rotating speed, and fermentation 2-3 days, obtains liquid bacterial suspension; Two kinds of bacterial suspensions, after concentrating low-temperature is dry, are prepared into conidial powder, containing living bacteria count >=10,000,000,000/gram;
3) preparation of fungal spore powder
Bacterial strain being cultivated in the pythium oligandrum inclined-plane that activated, be inoculated in level liquid seed culture medium, is to cultivate on the shaking table of 200 revs/min at 25-28 ℃ of temperature, rotating speed, fermentation 2-3 days; Then being transferred in secondary seed shaking flask, is to cultivate on the shaking table of 200 revs/min at 25-28 ℃ of temperature, rotating speed, and fermentation 2-3 days, then be transferred in bacterial fermentation tank medium is to cultivate on the shaking table of 200 revs/min at 25-28 ℃ of temperature, rotating speed, fermentation 2-3 days; The bacterial classification of bacterial fermentation tank medium is inoculated in solid fermentation medium, fermentation 3-5 days, form a large amount of spores, spore-bearing solid fermentation medium is infiltrated in sterile water, prepare spore suspension, then fungal spore suspension is concentrated, low temperature drying, makes fungal spore powder, spore count>=1 * 10
9individual/g;
4) complex microorganism bactericide product preparation
After three kinds of conidial powder that step 2 and 3 is obtained mix in the ratio of 1:1:1, it is former semen multiplex powder; According to following ratio, make complex microorganism bactericide product, former semen multiplex powder 3%, the nutrients of cooperation 5%-10%, 5% wetting agent, 15% dispersant, the carrier material of 65%-75%.
5. the preparation method of wide spectrum complex microorganism bactericide according to claim 4, is characterized in that: in step 2 and step 3, the inoculum concentration of bacillus thuringiensis and bacillus amyloliquefaciens and pythium oligandrum inclined-plane cultivation bacterial strain is 5%-10%.
6. the preparation method of wide spectrum complex microorganism bactericide according to claim 5, is characterized in that: in step 2, the fermentation time of bacillus thuringiensis and two kinds of bacterial strains of bacillus amyloliquefaciens is 2 days.
7. the preparation method of wide spectrum complex microorganism bactericide according to claim 6, is characterized in that: in step 1, bacterial classification brings back to life culture medium prescription: potato 20%, glucose 2%, beef extract 0.3%, peptone 1%, sodium chloride 0.5%, agar 1.5%, concrete making: get a certain amount of potato, remove the peel, be cut into piece and boil 30min, then use 200 order filtered through gauze, add again above-mentioned other raw materials to supply water, supply water, packing test tube after agar dissolves, 121 ℃ of sterilizing 30min.
8. the preparation method of wide spectrum complex microorganism bactericide according to claim 7, it is characterized in that: liquid seed culture medium formula in step 2: potato 20%, glucose 2%, beef extract 0.3%, peptone 1%, sodium chloride 0.5%, concrete making: get the potato of 200g peeling, be cut into piece and boil 30min, then use 200 order filtered through gauze, add again above-mentioned other raw materials to supply water, 121 ℃ of sterilizing 30min.
9. the preparation method of wide spectrum complex microorganism bactericide according to claim 8, it is characterized in that: the bacterial fermentation tank medium in step 2 and 3 forms and mass content is: molasses 3-5%, beef extract 0.3%, peptone 1%, sodium chloride 0.5%, magnesium sulfate 0.05%, potassium dihydrogen phosphate 0.05%, potassium nitrate 0.1%, adds water and supplies, and pH value is 6.8-7.4.
10. the preparation method of wide spectrum complex microorganism bactericide according to claim 9, it is characterized in that: solid fermentation culture medium prescription in step 2: soya-bean cake 5%, corn flour 10%, wheat bran 50%, powder of straw 35%, wherein the mass ratio of solid material and moisture is 3:2.
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