CN1568709A - Composite fungus agent for preventing blight of vegetable in greenhouse - Google Patents
Composite fungus agent for preventing blight of vegetable in greenhouse Download PDFInfo
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- CN1568709A CN1568709A CN 200410014880 CN200410014880A CN1568709A CN 1568709 A CN1568709 A CN 1568709A CN 200410014880 CN200410014880 CN 200410014880 CN 200410014880 A CN200410014880 A CN 200410014880A CN 1568709 A CN1568709 A CN 1568709A
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Abstract
A biological control bacteria formulation for preventing green house vegetable disease comprises Bacillus circulans, Bacillus pumilus, Bacillus cereus, Bacillus Subtilis, Serratia marcescens, Pseudomonas fluorescens. The preparation method is : mixing the above-mentioned bacteria according to 1:0.4-0.5:0.2-0.3:0.2-0.3:0.2-0.3:0.2-0.3, 200rpm shaking 36 hours in PDA culture liquid in 33 DEG C, 7000rpm centrifuging, preparing formulation using mixed bacteria and bacteria preservative fluid according to 1:40(g/ml), the alive bacteria concentration being 1x10<9>-1x10<14>cfu/ml. The bacteria formulation can be used in dunghill, root irrigation while transplanting after dilution. The preventing effect of bacteria formulation to vegetable disease can reach 75%, yield promoting effect is more than 45%.
Description
One, technical field
The composite bacteria agent capable (PGPR microbial inoculum) of control curing glasshouse vegetable epidemic disease belongs to the crops technical field of preventing and curing diseases, and is exclusively used in the non-environmental pollution control of vegetable epidemic disease.
Two, technical background
The cultivated area of the ground of China's protection at present vegetables constantly enlarges, owing to protect the suitable mould growth of epidemic disease of temperature and humidity condition in the ground, adds the continuous cropping of same kind, causes eqpidemic disease to increase the weight of year by year.The chemical control to vegetable epidemic disease at present mainly also is to use chemical pesticide, as: aliette, metalaxyl, Pu Like, Amici reach etc.But, use chemical pesticide not only can produce residue of pesticide, and along with the pesticide resistance of using phytophthora year by year of chemical pesticide is also increasing gradually, the Phytophthora capsici bacterial strain of resistance occurred Ridmil gold is had as Michigan, USA, and discovered when a bacterial strain develops immunity to drugs to Ridmil gold and can not lose original other characteristics simultaneously.(M.Hausbeck?R.Fulbright?and?R.Hammerschmidt.An?integrated?approachto?manage?phytophthora?blight?on?Michigan′s?vine?crops.)。The vegetable grower can only strengthen the usage amount of agricultural chemicals for this reason, and thus, agricultural chemicals uses and degree of dependence presents the vicious circle state.A large amount of uses of agricultural chemicals make in the vegetables that the persticide residue problem that exceeds standard is outstanding.In May, 2000, The Institute for the Control of Agrochemicals of the Ministry of Agriculture,PRC organized the agriculture chemical examination institution of 5 provinces and cities such as Beijing, Shanghai, Chongqing, Shandong and Zhejiang, to 50 vegetable varieties, the residue of pesticide of 1293 samples are carried out sampling Detection, the persticide residue exceeding standard rate reaches 30%, and the high person of residual concentration is several times even tens times of the permission residual quantity.The severe overweight of persticide residue in the vegetables causes intoxication accident to happen occasionally.According to Ministry of Public Health's statistics, China in 1999 is because food supply retail shop's dish food poisoning dish that residue of pesticide cause has 37, and the example of acute poisoning can also cause our attention, and the situation of chronic poisoning and retention toxicosis we are just unknown, its result can be more fearful in fact.China exports to the vegetables of developed countries such as Japan, Korea S and is not often return because residue of pesticide do not reach their standard, and this causes very big loss for many vegetable growers.Should widely popularize the plantation pollution-free vegetable, the standard production standard reduces the consumption of chemical pesticide as far as possible for this reason.
Pollution-free vegetable becomes the main flow that world vegetables produce already.As far back as the twenties in 20th century, just begin to develop pollution-free vegetable abroad, its main mode of production is soilless culture.According to incompletely statistics, single in the world country with NFT (NFT) cultivation pollution-free vegetable just reaches 76.In New Zealand, fruit vegetableses such as tomato more than half, cucumber are soilless cultures.Developed countries such as Japan, Holland, the U.S. adopt modern water planting greenhouse, produce pollution-free vegetable throughout the year.Research and the production of China pollution-free vegetable start from nineteen eighty-two, hold national biological control meeting this year, and Jiangsu Province takes the lead in proposing to replace chemical pesticide control with biological control.Nineteen eighty-three, under the support energetically of national plant protection master station, the whole nation 23 provinces, cities have carried out research, demonstration and the popularization of pollution-free vegetable.Logical research practice is in a few years explored a pollution-free vegetable production art of overlapping integrated control damage by disease and insect, minimizing pesticide pollution.The whole nation in 1985 is promoted pollution-free vegetable and is produced area 40,000 hm2 (600,000 mu), and almost all there is the pollution-free vegetable production base all parts of the country in each city so far.During this time, developed some successful biological bactericides, as agricultural streptomycin, jinggangmycin, the former can be made into the 200mg/L liquid irrigating root and prevent and treat vegetable bacterial soft rot, bacterial wilt, and the latter is made into 1000-1500 times of liquid control vegetables anthracnose, downy mildew.But the biological prevention and control agent of higher preventive effect does not appear eqpidemic disease is had as yet.
Three, summary of the invention
Technical problem does not have the biological prevention and control agent present situation of higher preventive effect at the greenhouse eqpidemic disease, provides exploitation a kind of composite bacteria agent capable of preventing and treating vegetable epidemic disease, is exclusively used in control cucumber, tomato, capsicum isothermal chamber vegetable epidemic disease, the preventive effect height of microbial inoculum, and the volume increase function is arranged.
Technical scheme
A kind of composite bacteria agent capable of preventing and treating curing glasshouse vegetable epidemic disease of the present invention, the main bacteria bacterial strain that comprises is:
Bacillus circulans (Bacillus circulans) bacterial strain B130, bacillus pumilus (B.pumilus) bacterial strain FH17, wax shape bacillus (B.cereus) the bacterial strain BB11 that stakes on, bacillus subtilis (B.subtilis) bacterial strain J1, serratia marcescens (Serratia marcescens) bacterial strain J2, pseudomonas fluorescens (Pseudomonas fluorescens) bacterial strain J3, the mixed proportion of each bacterial strain is: 1: 0.4～0.5: 0.2～0.3: 0.2～0.3: 0.2～0.3: 0.2～0.3.
The mixed culturing method of each bacterium is:
1) each bacterium is inoculated into cultivates 33 ℃ of 200rpm in the PDA culture fluid respectively simultaneously, after 24 hours in superclean bench the sampling OD value of surveying the 600nm place respectively, return to zero with the culture fluid that does not connect bacterium in the mensuration process;
2) each bacterium is cultured to the OD value all between 0.1～1 the time, begins to mix bacterium: get 1 milliliter of the bacterium of OD value maximum, then the OD value of proportioning value/this bacterium of the amount of other certain bacterium (ml)=maximum OD value * this bacterium and maximum OD value bacterium;
3) 33 ℃ of 200rpm shook 36 hours in the 250ml PDA culture fluid with being added to after each bacterium mixing, and 7000rpm is centrifugal then, and every 1000ml culture fluid obtains the wet bacterium of 13～16g, and the wet bacterium of every gram contains 4 * 10
10～10
15Individual thalline.
Get wet bacterium and preservative fluid for thallus and be made into microbial inoculum by 1: 40 (g/ml), the viable bacteria total concentration is 1 * 10 in the finished product
9～1 * 10
14Cfu/ml is used to prevent and treat curing glasshouse vegetable epidemic disease.Wherein used PDA culture fluid compound method is: be cut into small pieces to be put in the water after peeling with 200 gram potatoes and boil, at least boil more than 20 minutes after the boiling, then filter with four layers of hospital gauze, filtrate adds 20 gram common sucrose, be settled to 1000 milliliters, pH value transfers to 7.2-7.4, sterilizes 20 minutes for 121 ℃ then.
Beneficial effect
The present invention compares with existing vegetable epidemic disease Prevention Technique, and its advantage and good effect show:
The present invention is specially at the biological prevention and control agent of vegetable epidemic disease exploitation, thereby significantly improves comparing control efficiency with other broad spectrum biological disinfection agent at present aspect the control vegetable epidemic disease.Owing to be biologic product, do not have a series of problems that use brought because of chemical pesticide fully, thereby help the nuisanceless production of vegetables, the peasant can or reduce other consumptions of preventing and treating the eqpidemic disease chemical pesticide, this not only can be the peasant and reduces expenses, and helps the outlet of vegetables.Simultaneously, this microbial inoculum increases production function in addition, can be the peasant and increases income.
This microbial inoculum with wet bacterium and this breadboard preservative fluid for thallus (belong to this laboratory granted patent, patent No. ZL02112559.7, publication CN1358838, open day: on July 17th, 2002) be made into microbial inoculum by 1: 40 (g/ml), but room temperature preservation 2～3 years.
This microbial inoculum can be poured into before transplant in the greenhouse and make compost in the fertilizer, buries in the soil in whole ground process with fertilizer then, also can irritate root and use when planting seedling.After planting seedling, bacterium wherein can be at the root colonization of seedling, and some can produce antagonistic substance and destroy the mould cell membrane of epidemic disease, the interchange between organelle, and the degradation of cell wall destroys the chlamydospore of phytophthora etc.; Other then occupy the rhizosphere space, and with phytophthora contention nutrition, as the secretion of root etc., thereby reach the effect of preventing and treating eqpidemic disease.
Test shows, uses this microbial inoculum can reach more than 75% the average control efficiency of vegetable epidemic disease, and volume increase can reach more than 45%.
Four embodiments
A kind of composite bacteria agent capable (commodity are called the PGPR composite bacteria agent capable) of vegetable epidemic disease of preventing and treating of the present invention is by bacterial strain B130 (Bacillus circulans Bacillus circulans, Journal of Bacteriology, July1990, p.4017-4022), FH17 (B.pumilus bacillus pumilus, Phytopathology Vol.92, No.12:1329-1333,2002), BB11 (the B.cereus wax shape bacillus of staking on, Appl.Environ.Microbol.69:6208-6215; 2003), J1 (B.subtilis bacillus subtilis, Plant Physiology Jan2004.Vol.134, Iss.1; P.307), J2Serratia marcescens (serratia marcescens, Annals of the phytopathological society of Japan1998,64:4,287-293), J3 (Pseudomonasfluorescens pseudomonas fluorescens, Appl.Environ.Microbol.Vol65, No.6.2429-2438; 1999) etc. the known public bacterial strain of people and animals and plants no pathogenicity is formed.
Wherein bacterial strain B130 sees document: the Primary Study of the anti-blue or green withered biocontrol microorganisms antagonist character of .1994. such as Guo Jianhua. the journal .18 of Agricultural University Of Nanjing (2): 59 ∽ 62.; All the other bacterial strain FH17,, BB11, J1, J2, J3 all see document: the screening of .2001. pepper ralstonia solanacearum antagonistic strains such as Guo Jianhua and the mensuration of field efficacy. Chinese biological control .17 (3): 101 ∽ 106.
More than each bacterial strain belong to public bacterial strain commonly used, (laboratory, inventor place) all has preservation in Agricultural University Of Nanjing plant pathology laboratory, can guarantee to supply to society for a long time.
This microbial inoculum adopts conventional bacterial cultivation to obtain, above each bacterium is mixed the back in 1: 0.4～0.5: 0.2～0.3: 0.2～0.3: 0.2～0.3: 0.2～0.3 ratio to shake 36 hours with 33 ℃ of 200rpm of PDA culture fluid, 7000rpm is centrifugal then, every 1000ml culture fluid obtains the wet bacterium of 13～16g, and the wet bacterium of every gram contains 4 * 10
10～10
15Individual thalline.
Get wet bacterium and be made into microbial inoculum with this breadboard preservative fluid for thallus (belonging to this laboratory granted patent, patent No. ZL02112559.7, publication CN1358838) by 1: 40 (g/ml), the viable bacteria total concentration is 1 * 10 in the finished product
9～1 * 10
14Cfu/ml.
PDA culture fluid compound method: be cut into small pieces to be put in the water after peeling with 200 gram potatoes and boil, boil at least after the boiling more than 20 minutes, then with four layers of hospital gauzes filtration, filtrate adds 20 gram common sucrose, be settled to 1000 milliliters, pH value transfers to 7.2-7.4, sterilizes 20 minutes for 121 ℃ then.
Each bacterium mixed method: each bacterium is inoculated into cultivates 33 ℃ of 200rpm in the PDA culture fluid respectively simultaneously, in superclean bench, take a sample respectively after about 24 hours and survey the OD value at 600nm place, return to zero with the culture fluid that does not connect bacterium in the mensuration process.When the OD value is not between 0.1～1, each bacterium is continued to cultivate, surveyed once every 3 hours, up to each OD value all in this scope.Can begin this moment to mix bacterium, for example get 1 milliliter of the maximum bacterium of OD value, then the OD value of proportioning value/this bacterium of the amount of other certain bacterium (ml)=maximum OD value * this bacterium and maximum OD value bacterium.Annotate: it has been generally acknowledged that when the OD of 600nm place value is between 0.1～1, to meet concentration=OD value * 10
9Cfu/ml.33 ℃ of 200rpm shook 36 hours in the 250ml PDA culture fluid with being added to after each bacterium mixing, and common every 1000ml culture fluid can be produced the wet bacterium of 13～16g).
This microbial inoculum can be poured into before transplant in the greenhouse and make compost in the fertilizer, buries in the soil in whole ground process with fertilizer then, also can irritate root and use when planting seedling.After planting seedling, bacterium wherein can be at the root colonization of seedling, and some can produce antagonistic substance and destroy the mould cell membrane of epidemic disease, the interchange between organelle, and the degradation of cell wall destroys the chlamydospore of phytophthora etc.; Other then occupy the rhizosphere space, and with phytophthora contention nutrition, as the secretion of root etc., thereby reach the effect of preventing and treating eqpidemic disease.
Embodiment 1: gets by bacterial strain B130, and FH17, BB11, J1, J2, the J3 mixed proportion is 1: 0.5: 0.2: 0.3: 0.3: 0.2 wet bacterium, the wet bacterium of every gram contains 4 * 10
10Individual thalline, add preservative fluid for thallus sodium borate buffer liquid, to final concentration be 0.01mol/L (pH7.3), add Tween20 to final concentration be 0.001mol/L, promptly obtain composite bacteria agent capable (commodity are called the PGPR composite bacteria agent capable), but room temperature preservation 2～3 years, be used for capsicum epidemic disease biological and ecological methods to prevent plant disease, pests, and erosion experiment (table 1), the result shows, uses this microbial inoculum can reach 81.3% to the average control efficiency of vegetable epidemic disease, volume increase can reach 96.4%, apparently higher than control group and above each bacterial strain list bacteria preparation.
The capsicum epidemic disease biological and ecological methods to prevent plant disease, pests, and erosion experiment of Ganyu, table 1 Jiangsu in 2003
| Processing scheme | The incidence of disease (%) | Preventive effect (%) | Rate of growth (%) | |
| Control group | The 50kg fertilizer is directly used | 12 | ||
| Experimental group a | 4000ml composite bacteria agent capable/mu compost treatment | 2.3 | ?81.3 | ?96.4 |
| Experimental group b | 2000ml composite bacteria agent capable/mu compost treatment | 7 | ?41.7 | ?62.5 |
| Experimental group c | 1000ml composite bacteria agent capable/mu compost treatment | 10.6 | ?12 | ?34.7 |
| B.circulans?B130 | The single bacteria preparation of 4000ml/mu compost treatment | 9.4 | ?21.3 | ?18.7 |
| B.pumilus?FH17 | The single bacteria preparation of 4000ml/mu compost treatment | 8.9 | ?25.6 | ?14.3 |
| ?B.cereus?BB11 | The single bacteria preparation of 4000ml/mu compost treatment | 9.2 | ?23.2 | ?13 |
| ?B.subtilis?J1 | The single bacteria preparation of 4000ml/mu compost treatment | 8.0 | ?33.4 | ?10.6 |
| ?S.marcescens?J2 | The single bacteria preparation of 4000ml/mu compost treatment | 6.5 | ?45.7 | ?11.5 |
| ?P.fluorescens?J3 | The single bacteria preparation of 4000ml/mu compost treatment | 7.5 | ?37.5 | ?16.3 |
Embodiment 2: gets by bacterial strain B130, and FH17, BB11, J1, J2, the J3 mixed proportion is 1: 0.4: 0.2: the wet bacterium that obtains at 0.3: 0.3: 0.3, the wet bacterium of every gram contains 4 * 10
15Individual thalline adds preservative fluid for thallus sodium borate buffer liquid, to final concentration be 0.01mol/L (pH7.3), add Tween20 to final concentration be 0.001mol/L, promptly obtain composite bacteria agent capable (commodity are called the PGPR composite bacteria agent capable), room temperature preservation is used for eqpidemic disease biological and ecological methods to prevent plant disease, pests, and erosion experiment (table 2, table 3) for a long time, the result shows, use this microbial inoculum can reach 95.6% to the average control efficiency of cucumber eqpidemic disease, volume increase can reach 48.1%, apparently higher than control group and above each bacterial strain list bacteria preparation.
Fugou County Dongying village, table 2 Henan Province cucumber eqpidemic disease biological and ecological methods to prevent plant disease, pests, and erosion experiment (2000)
| Processing scheme | The incidence of disease (%) | Preventive effect (%) | Rate of growth (%) | |
| Control group | Transplanting and each time are later on all watered | 22.9 | ||
| Experimental group | Composite bacteria agent capable scheme 1 is handled | 1.2 | ?95.6 | ?48.1 |
| B.circulans?B130 | Single bacteria preparation scheme 1 is handled | 14.7 | ?35.6 | ?15.3 |
| B.pumilus?FH17 | Single bacteria preparation scheme 1 is handled | 13.7 | ?40.2 | ?15.6 |
| B.cereus?BB11 | Single bacteria preparation scheme 1 is handled | 14.9 | ?35.1 | ?14.3 |
| B.subtilis?J1 | Single bacteria preparation scheme 1 is handled | 15.3 | ?33.2 | ?18.6 |
| S.marcescens?J2 | Single bacteria preparation scheme 1 is handled | 13.6 | ?40.6 | ?10.2 |
| P.fluorescens?J3 | Single bacteria preparation scheme 1 is handled | 12.5 | ?45.3 | ?14.1 |
Annotate: 1000 times of scheme 1 microbial inoculum dilutions, mixing is irritated root as normal root water when transplanting, and the about 50ml of every strain is advisable to irritate enough normal root water, and this waters once for another example after the week; Used once every 30 days later on, use altogether 7 times.
Fugou County Dongying village, table 3 Henan Province cucumber eqpidemic disease biological and ecological methods to prevent plant disease, pests, and erosion experiment (calendar year 2001)
| Processing scheme | The incidence of disease (%) | Preventive effect (%) | Rate of growth (%) | |
| Control group | Transplanting and each time are later on all watered | 17.6 | ||
| Experimental group | Composite bacteria agent capable scheme 1 is handled | 2.7 | 87.7 | ?53.8 |
Embodiment 3: gets by bacterial strain B130, and FH17, BB11, J1, J2, the J3 mixed proportion is 1: 0.4: 0.3: the wet bacterium that obtains at 0.2: 0.2: 0.3, the wet bacterium of every gram contains 4 * 10
14Individual thalline adds preservative fluid for thallus sodium borate buffer liquid, to final concentration be 0.01mol/L (pH7.3), add Tween20 to final concentration be 0.001mol/L, promptly obtain composite bacteria agent capable, room temperature preservation is used for capsicum epidemic disease biological and ecological methods to prevent plant disease, pests, and erosion experiment (table 4, table 5) for a long time, the result shows, use this microbial inoculum can reach 87.8% to the average control efficiency of cucumber eqpidemic disease, volume increase can reach 247.5%, apparently higher than control group and above each bacterial strain list bacteria preparation.
Handan, table 4 Hebei cucumber eqpidemic disease biological and ecological methods to prevent plant disease, pests, and erosion experiment (2000)
| Processing scheme | The incidence of disease (%) | Preventive effect (%) | Rate of growth (%) | |
| Control group | Transplanting and each time are later on all watered | 84.7 | ||
| Experimental group | Composite bacteria agent capable scheme 1 is handled | 13.4 | ?87.8 | ?247.5 |
| ?B.circulans?B130 | Single bacteria preparation scheme 1 is handled | 46.3 | ?45.3 | ?15.3 |
| ?B.pumilus?FH17 | Single bacteria preparation scheme 1 is handled | 52.9 | ?37.5 | ?15.4 |
| ?B.cereus?BB11 | Single bacteria preparation scheme 1 is handled | 50.3 | ?40.6 | ?14.2 |
| ?B.subtilis?J1 | Single bacteria preparation scheme 1 is handled | 47.2 | ?44.2 | ?13.9 |
| ?S.marcescens?J2 | Single bacteria preparation scheme 1 is handled | 43.5 | ?48.6 | ?14.7 |
| ?P.fluorescens?J3 | Single bacteria preparation scheme 1 is handled | 37.9 | ?55.2 | ?16.2 |
Handan, table 5 Hebei cucumber eqpidemic disease biological and ecological methods to prevent plant disease, pests, and erosion experiment (calendar year 2001)
| Processing scheme | The incidence of disease (%) | Preventive effect (%) | Rate of growth (%) | |
| Control group | Transplanting and each time are later on all watered | 81.5 | ||
| Experimental group | Composite bacteria agent capable scheme 1 is handled | 12.4 | 87.9 | 239.6 |
Embodiment 4: gets by bacterial strain B130, and FH17, BB11, J1, J2, the J3 mixed proportion is 1: 0.5: 0.2: the wet bacterium that obtains at 0.3: 0.2: 0.2, the wet bacterium of every gram contains 4 * 10
13Individual thalline, add preservative fluid for thallus sodium borate buffer liquid, to final concentration be 0.01mol/L (pH7.3), add Tween20 to final concentration be 0.001mol/L, promptly obtain composite bacteria agent capable, room temperature preservation for a long time, be used for capsicum epidemic disease biological and ecological methods to prevent plant disease, pests, and erosion experiment (table 6), the result shows, uses this microbial inoculum can reach 78.7% to the average control efficiency of tomato blight, volume increase can reach 48.6%, apparently higher than control group and above each bacterial strain list bacteria preparation.
Table 6 Nanjing herds of horses tomato blight biological and ecological methods to prevent plant disease, pests, and erosion experiment (1999)
| Processing scheme | The incidence of disease (%) | Preventive effect (%) | Rate of growth (%) | |
| Control group | Direct application of organic fertilizers 100kg | 47 | ||
| Experimental group | 4000ml composite bacteria agent capable/mu compost | 10 | ?78.7 | ?48.6 |
| ?B.circulans?B130 | The single bacteria preparation of 4000ml/mu compost | 31.7 | ?32.6 | ?15.3 |
| ?B.pumilus?FH17 | The single bacteria preparation of 4000ml/mu compost | 31.4 | ?33.1 | ?18.9 |
| ?B.cereus?BB11 | The single bacteria preparation of 4000ml/mu compost | 32.7 | ?30.4 | ?14.3 |
| ?B.subtilis?J1 | The single bacteria preparation of 4000ml/mu compost | 27.9 | ?40.6 | ?20.5 |
| ?S.marcescens?J2 | The single bacteria preparation of 4000ml/mu compost | 25.7 | ?45.2 | ?12.3 |
| ?P.fluorescens?J3 | The single bacteria preparation of 4000ml/mu compost | 28.8 | ?38.7 | ?14.7 |
Claims (6)
1, a kind of composite bacteria agent capable of preventing and treating curing glasshouse vegetable epidemic disease is characterized in that, the main bacteria bacterial strain that comprises is:
Bacillus circulans (Bacillus circulans) bacterial strain B130, bacillus pumilus (B.pumilus) bacterial strain FH17, wax shape bacillus (B.cereus) the bacterial strain BB11 that stakes on, bacillus subtilis (B.subtilis) bacterial strain J1, serratia marcescens (Serratia marcescens) bacterial strain J2, pseudomonas fluorescens (Pseudomonas fluorescens) bacterial strain J3, the mixed proportion of each bacterial strain is: 1: 0.4～0.5: 0.2～0.3: 0.2～0.3: 0.2～0.3: 0.2～0.3.
2, a kind of composite bacteria agent capable of preventing and treating curing glasshouse vegetable epidemic disease according to claim 1 is characterized in that the mixed culturing method of each bacterium is:
1) each bacterium is inoculated into cultivates 33 ℃ of 200rpm in the PDA culture fluid respectively simultaneously, after 24 hours in superclean bench the sampling OD value of surveying the 600nm place respectively, return to zero with the culture fluid that does not connect bacterium in the mensuration process;
2) each bacterium is cultured to the OD value all between 0.1～1 the time, begins to mix bacterium: get 1 milliliter of the bacterium of OD value maximum, then the OD value of proportioning value/this bacterium of the amount of other certain bacterium (ml)=maximum OD value * this bacterium and maximum OD value bacterium;
3) 33 ℃ of 200rpm shook 36 hours in the 250ml PDA culture fluid with being added to after each bacterium mixing, and 7000rpm is centrifugal then, and every 1000ml culture fluid obtains the wet bacterium of 13～16g, and the wet bacterium of every gram contains 4 * 10
10～10
15Individual thalline.
3, a kind of composite bacteria agent capable of preventing and treating curing glasshouse vegetable epidemic disease according to claim 1 and 2 is characterized in that, used PDA culture fluid compound method is:
Be cut into small pieces to be put in the water after peeling with 200 gram potatoes and boil, boil at least after the boiling more than 20 minutes, then filter with four layers of hospital gauze, filtrate adds 20 gram common sucrose, is settled to 1000 milliliters, and pH value transfers to 7.2-7.4, sterilizes 20 minutes for 121 ℃ then.
According to claim 1 or 2 described a kind of composite bacteria agent capables of preventing and treating curing glasshouse vegetable epidemic disease, it is characterized in that 4, get wet bacterium and preservative fluid for thallus and be made into microbial inoculum by 1: 40 (g/ml), the viable bacteria total concentration is 1 * 10 in the finished product
9～1 * 10
14Cfu/ml.
According to the described a kind of composite bacteria agent capable of preventing and treating curing glasshouse vegetable epidemic disease of claim 3, it is characterized in that 5, get wet bacterium and preservative fluid for thallus and be made into microbial inoculum by 1: 40 (g/ml), the viable bacteria total concentration is 1 * 10 in the finished product
9～1 * 10
14Cfu/ml.
6, the described a kind of application that prevents and treats the composite bacteria agent capable of curing glasshouse vegetable epidemic disease of one of claim 1-5.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101381761B (en) * | 2008-10-17 | 2010-10-27 | 山东省农业科学院土壤肥料研究所 | Application of duel functional composite bacteria agent capable of decomposition of stalk and low-temperature production of biogas |
| CN101643711B (en) * | 2009-05-31 | 2012-05-23 | 南京林业大学 | Bacillus pumilus and application in preventing and treating dothiorella gregaria |
| CN103109870A (en) * | 2013-01-08 | 2013-05-22 | 华中农业大学 | Application of fermentation supernatant liquid of pseudomonas |
| CN103146609A (en) * | 2013-03-08 | 2013-06-12 | 南京师范大学 | Pseudomonas fluorescens and method for preventing phytophthora capsici thereby |
| CN103215203A (en) * | 2013-03-25 | 2013-07-24 | 南京农业大学 | Biocontrol strain BF 6 for preventing and treating tomato yellow leaf curl virus and application thereof |
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Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1032840C (en) * | 1993-02-19 | 1996-09-25 | 北京农业大学 | Production-increasing compounded bacterium and its production method |
| CN1040719C (en) * | 1994-05-18 | 1998-11-18 | 中国科学院新疆生物土壤沙漠研究所 | PG6 micro-organism preparation capable of raising yield of wheat, barley, corn et al. crops |
| CN1120236C (en) * | 2000-12-14 | 2003-09-03 | 中国科学院新疆生态与地理研究所 | PR3 bacteria preparation for resisting phytophthora disease |
| CN1162536C (en) * | 2002-01-18 | 2004-08-18 | 南京农业大学 | Preservation method for live bacterial preparation |
| CN1176878C (en) * | 2002-01-18 | 2004-11-24 | 南京农业大学 | Composit method for preventing plant soil spread diseases |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101381761B (en) * | 2008-10-17 | 2010-10-27 | 山东省农业科学院土壤肥料研究所 | Application of duel functional composite bacteria agent capable of decomposition of stalk and low-temperature production of biogas |
| CN101643711B (en) * | 2009-05-31 | 2012-05-23 | 南京林业大学 | Bacillus pumilus and application in preventing and treating dothiorella gregaria |
| CN103109870A (en) * | 2013-01-08 | 2013-05-22 | 华中农业大学 | Application of fermentation supernatant liquid of pseudomonas |
| CN103109870B (en) * | 2013-01-08 | 2014-06-11 | 华中农业大学 | Application of fermentation supernatant liquid of pseudomonas |
| CN103146609A (en) * | 2013-03-08 | 2013-06-12 | 南京师范大学 | Pseudomonas fluorescens and method for preventing phytophthora capsici thereby |
| CN103215203A (en) * | 2013-03-25 | 2013-07-24 | 南京农业大学 | Biocontrol strain BF 6 for preventing and treating tomato yellow leaf curl virus and application thereof |
| CN103215203B (en) * | 2013-03-25 | 2014-10-22 | 南京农业大学 | Biocontrol strain BF 6 for preventing and treating tomato yellow leaf curl virus and application thereof |
| CN105766308A (en) * | 2016-03-15 | 2016-07-20 | 成都繁尚农业有限公司 | Method for high yield of organic tomatoes |
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