CN107410366A - A kind of long-acting biological kills the preparation method of nematode combination agent - Google Patents
A kind of long-acting biological kills the preparation method of nematode combination agent Download PDFInfo
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Abstract
A kind of long-acting biological kills the preparation method of nematode combination agent, including：Step (1), Metarhizium anisopliae (Metarhizium guizhouense) strain of Cord blood is subjected to seed culture；Step (2), Shaking culture strain；Step (3), initial fermentation；Step (4), depth fermentation；Step (5), prepares bacteria suspension；Step (6), the bacteria suspension that step (5) obtains is well mixed with athomin, Bravo, citric acid and nimbin, is drying to obtain described long-acting biological nematicidal composition.Present invention employs the Metarhizium anisopliae with excellent nematicide ability, and in preparation process, its bacterium solution is adsorbed on activated carbon, part bacterium solution is adsorbed in inside activated carbon duct, is slowly discharged after so that the present invention possesses long-acting nematocidal effect.
Description
Technical field
The present invention relates to agriculture nematicide agent preparation field, and in particular to a kind of long-acting biological kills the system of nematode combination agent
Preparation Method.
Background technology
In Agricultural Activities, agricultural crops are often subject to disease, cause a large amount of underproduction.The harm of plant nematode
More than bacterium and virus, fungal disease is only second to.Plant nematode causes the loss about 100,000,000,000 that world agriculture produces every year
Dollar, wherein root-knot nematode is the maximum a kind of plant nematode of harm, is often only and industrial crops important in the world are made
Into economic loss just be up to tens billion of dollars.Nematode is to most cereal crops, oil crops, fibre crops, tobacco, tea
Leaf, fruit tree, vegetables, medicinal material and flowers etc. can cause heavy losses.
In the method for control nematodiasis, because root-knot nematode is generally survived in soil and in plant roots, so one
As chemical pesticide be difficult to control its harm, highly toxic pesticide constitutes a serious threat to the security of agricultural product production again, and chemistry kills
Nematode agent can only control nematode population in a short time, while the problems such as short cost height, holding effect and resistance to the action of a drug also be present.In addition, one
A little traditional prevention and controls, though such as crop rotation, plantation disease-resistant variety can play certain preventive and therapeutic effect, the energy in actual production
The crop of enough crop rotations is simultaneously few, and the crop varieties resistant to root-knot nematode are extremely limited.
Microbial insecticide has to person poultry safety, harmless to natural enemy insect, selectivity is high, effect on environment is small, is not easy
The features such as developing immunity to drugs and more and more paid attention to.But the action effect of GR of the prior art is short
Phase is more obvious, over time, kills nematode ability almost without depositing.
The content of the invention
The purpose of the present invention is in view of the shortcomings of the prior art, there is provided a kind of long-acting biological kills nematode combination agent.The present invention
Long-acting biological kill nematode combination agent and have and efficiently kill nematode, and the advantages of continuous and effective.
Technical scheme is as follows.
A kind of long-acting biological kills the preparation method of nematode combination agent, including：
Step (1), by the Metarhizium anisopliae (Metarhizium guizhouense) of Cord blood, strain transfer is extremely
On PDA culture medium inclined-plane, cultivated 2-4 days under the conditions of 25-35 DEG C, realize the abundant activation of strain, the strain after activation culture connects
Kind shaking flask carries out seed culture；
Step (2), Shake flask medium is selected, consisting of glucose 1-5 parts by weight, MgSO40.1-0.6 parts by weight,
KCl 0.1-0.3 parts by weight, ZnSO40.2-0.3 parts by weight, K2HPO40.1-0.9 parts by weight, agar 1-3 parts by weight, K2HPO4
0.1-0.9 parts by weight and distilled water 20-50 parts by weight；
Shaking flask is directly inoculated with using culture medium when shaking flask is inoculated with, the parameter of Shaking culture is as follows：Shaking speed is 150rpm,
Cultivation temperature is 29-30 DEG C, time 48-70h；Produce strain；
Step (3), prepares initial fermentation fluid nutrient medium, and the initial fermentation fluid nutrient medium includes：Glucose 10-15
Parts by weight, yeast 1-6 parts by weight, MgSO41-5 parts by weight, KCl 0.2-2 parts by weight, ZnSO40.2-3 parts by weight, distilled water
200-2000 parts by weight；
Seeding tank is sterilized and carries out initial fermentation, inoculation condition is：121 DEG C of culture medium sterilizing 30min, now pH scopes exist
6.0-6.5；Zymotic fluid is inoculated with by 4% volume ratio of canned amount, and inoculation temperature is less than 30 DEG C, and fermentation time is 18-24 hours；
Seed liquor is in thick obtained by initial fermentation, and spore content reaches 6 × 107/ more than ml；
Step (4), prepares depth fermentation broth, and the depth fermentation broth includes glucose 20-50
Parts by weight, yeast 10-15 parts by weight, MgSO46-10 parts by weight, KCl 5-9 parts by weight, ZnSO43-7 parts by weight, crops straw
Stalk 50-200 parts by weight and distilled water 500-3000 parts by weight；
The production tank sterilizing of depth fermentation, inoculation condition are specially：Culture medium presses tank through 121 DEG C of sterilizing 30min, zymotic fluid
The 4% volume ratio inoculation of loading amount, inoculation temperature are less than 30 DEG C；28 ± 1 DEG C of fermentation temperature, fermentation period are 42-50 hours；
Liquid gives birth to spore content 5 × 10 in depth fermentation gained bacterium solution8/ more than ml；
Step (5), the bacterium solution obtained by step (4) is stirred in fermentation tank, disperse mycelia and spore, add bacterium solution
The activated carbon of weight 10-28% weight and bacterium solution weight 0.01-2% polysorbate60, stirring, it is standby to be prepared into bacteria suspension；
Step (6), the bacteria suspension that step (5) is obtained is with athomin, Bravo, citric acid and nimbin according to 1-2:
0.1-0.8: 0.2-0.5: 0.01-0.2: 0.5-1.2 is well mixed, and is drying to obtain described long-acting biological nematicidal composition.
Preferably, in step (2), Shake flask medium composition is the parts by weight of glucose 2, MgSO40.5 parts by weight, KCl
0.2 parts by weight, ZnSO40.2 parts by weight, K2HPO40.6 parts by weight, the parts by weight of agar 2, K2HPO40.5 parts by weight and distilled water
30 parts by weight.
Preferably, in step (3), the initial fermentation fluid nutrient medium includes：The parts by weight of glucose 12, the weight of yeast 3
Part, MgSO43 parts by weight, the parts by weight of KCl 1, ZnSO41 parts by weight, the parts by weight of distilled water 1000.
Preferably, in step (4), the depth fermentation broth includes the parts by weight of glucose 30, the weight of yeast 12
Part, MgSO48 parts by weight, the parts by weight of KCl 6, ZnSO45 parts by weight, the parts by weight of agricultural crop straw 100 and the weight of distilled water 2000
Part.
Preferably, in step (6), bacteria suspension and athomin, Bravo, citric acid and nimbin that step (4) obtains
It is well mixed according to 1: 0.5: 0.4: 0.1: 0.8.
The present invention is further claimed a kind of long-acting biological and kills nematode combination agent, and its above-mentioned preparation method obtains.
A kind of described long-acting biological kills the application of nematode combination agent, is placed on crop root use.
The present invention achieves significant technological progress.
Present invention employs the Metarhizium anisopliae with excellent nematicide ability, and in preparation process, by its bacterium
Liquid is adsorbed on activated carbon, and part bacterium solution is adsorbed in inside activated carbon duct, is slowly discharged after so that the present invention possesses length
Imitate nematocidal effect.The present invention by with by the bacterium solution of Metarhizium anisopliae and athomin, Bravo, citric acid and nimbin
Compounding, overcomes the problem of function existing for single GR is single, raising kills nematode ability, alleviates nematode
The harm of disease, athomin produce escaping gas in decomposable process by its organic matter and eliminate or suppress to be harmful to life in soil
Thing, the inhibitory action, effect especially to root knot nematode disease are good, and nimbin can make root-knot nematode food refusal and suppress root-knot nematode growth
Development, disturbs root-knot nematode endocrine, and Bravo enables larva to be unable to normal growth and causes the dead citric acid added
There is provided above-mentioned nematicide the good pH environment to play a role, improve killing ability, in of the invention, Metarhizium anisopliae
Bacterium solution coordinates with athomin, Bravo, citric acid and nimbin to act synergistically, and rationally controls component ratio, and product is to line
The insecticidal effect of worm is extremely excellent, and cost is relatively low, and method is simple, friendly to environment and crop without any pollution, depth hair
Crop material has been used during ferment, has reduced goods cost.The present invention long-acting biological kill nematode combination agent have efficiently kill nematode, and
The advantages of continuous and effective.
Embodiment
Technical scheme is described in further detail with reference to embodiment.
Embodiment 1
A kind of long-acting biological kills the preparation method of nematode combination agent, including：
Step (1), by the Metarhizium anisopliae (Metarhizium guizhouense) of Cord blood, strain transfer is extremely
On PDA culture medium inclined-plane, cultivated 4 days under the conditions of 35 DEG C, realize the abundant activation of strain, the strain inoculation shaking flask after activation culture
Carry out seed culture；
Step (2), Shake flask medium is selected, consisting of the parts by weight of glucose 5, MgSO40.6 parts by weight, KCl 0.3
Parts by weight, ZnSO40.3 parts by weight, K2HPO40.9 parts by weight, the parts by weight of agar 3, K2HPO40.9 parts by weight and distilled water 50
Parts by weight；
Shaking flask is directly inoculated with using culture medium when shaking flask is inoculated with, the parameter of Shaking culture is as follows：Shaking speed is 150rpm,
Cultivation temperature is 30 DEG C, time 70h；Produce strain；
Step (3), prepares initial fermentation fluid nutrient medium, and the initial fermentation fluid nutrient medium includes：The weight of glucose 15
Measure part, the parts by weight of yeast 6, MgSO45 parts by weight, the parts by weight of KCl 2, ZnSO43 parts by weight, the parts by weight of distilled water 2000；
Seeding tank is sterilized and carries out initial fermentation, inoculum concentration and inoculation temperature are specially：121 DEG C of sterilizings 30min, now pH
Scope is 6.5；Zymotic fluid is inoculated with by 4% volume ratio of canned amount, and inoculation temperature is 30 DEG C, and fermentation time is 24 hours；Tentatively
Fermentation gained seed liquor is in thick, and spore content reaches 6 × 107/ more than ml；
Step (4), prepares depth fermentation broth, and the depth fermentation broth includes the weight of glucose 50
Part, the parts by weight of yeast 15, MgSO410 parts by weight, the parts by weight of KCl 9, ZnSO47 parts by weight, the parts by weight of agricultural crop straw 200 and
The parts by weight of distilled water 3000；
The production tank of depth fermentation sterilizes, inoculum concentration and inoculation temperature are specially：Nutrient solution connects through 121 DEG C of sterilizing 30min
Kind 4% volume ratio of amount, inoculation temperature are 30 DEG C；28 DEG C of fermentation temperature, fermentation period are 50 hours；
Liquid gives birth to spore content 5 × 10 in depth fermentation gained bacterium solution8/ more than ml, dry cell weight is 1.9%；
Step (5), the bacterium solution obtained by step (4) is stirred in fermentation tank, disperse mycelia and spore, add bacterium solution
The activated carbon of the weight of weight 28% and the polysorbate60 of bacterium solution weight 2%, it is standby to be prepared into bacteria suspension；
Step (6), the bacteria suspension that step (5) is obtained is with athomin, Bravo, citric acid and nimbin according to 2:
0.8: 0.5: 0.2: 1.2 is well mixed, is drying to obtain described long-acting biological nematicidal composition.
Embodiment 2
A kind of long-acting biological kills the preparation method of nematode combination agent, including：
Step (1), by the Metarhizium anisopliae (Metarhizium guizhouense) of Cord blood, strain transfer is extremely
On PDA culture medium inclined-plane, cultivated 2 days under the conditions of 25 DEG C, realize the abundant activation of strain, the strain inoculation shaking flask after activation culture
Carry out seed culture；
Step (2), Shake flask medium is selected, consisting of the parts by weight of glucose 1, MgSO40.1 parts by weight, KCl 0.1
Parts by weight, ZnSO40.2 parts by weight, K2HPO40.1 parts by weight, the parts by weight of agar 1, K2HPO40.1 parts by weight and distilled water 20
Parts by weight；
Shaking flask is directly inoculated with using culture medium when shaking flask is inoculated with, the parameter of Shaking culture is as follows：Shaking speed is 150rpm,
Cultivation temperature is 29 DEG C, time 48h；Produce strain；
Step (3), prepares initial fermentation fluid nutrient medium, and the initial fermentation fluid nutrient medium includes：The weight of glucose 10
Measure part, the parts by weight of yeast 1, MgSO41 parts by weight, the parts by weight of KCl 0.2, ZnSO40.2 parts by weight, the parts by weight of distilled water 200；
Seeding tank is sterilized and carries out initial fermentation, inoculum concentration and inoculation temperature are specially：121 DEG C of sterilizings 30min, now pH
Scope is 6.0；Zymotic fluid is inoculated with by 4% volume ratio of canned amount, and inoculation temperature is less than 30 DEG C, and fermentation time is 18 hours；
Seed liquor is in thick obtained by initial fermentation, and spore content reaches 6 × 107/ more than ml；
Step (4), prepares depth fermentation broth, and the depth fermentation broth includes the weight of glucose 20
Part, the parts by weight of yeast 10, MgSO46 parts by weight, the parts by weight of KCl 5, ZnSO43 parts by weight, the parts by weight of agricultural crop straw 50 and steaming
The parts by weight of distilled water 500；
The production tank of depth fermentation sterilizes, inoculum concentration and inoculation temperature are specially：Nutrient solution connects through 121 DEG C of sterilizing 30min
Kind 4% volume ratio of amount, inoculation temperature are 29 DEG C；28 DEG C of fermentation temperature, fermentation period are 42 hours；
Liquid gives birth to spore content 5 × 10 in depth fermentation gained bacterium solution8/ more than ml, dry cell weight is more than 1.8%；
Step (5), the bacterium solution obtained by step (4) is stirred in fermentation tank, disperse mycelia and spore, add bacterium solution
The activated carbon of the weight of weight 10% and the polysorbate60 of bacterium solution weight 0.01%, it is standby to be prepared into bacteria suspension；
Step (6), the bacteria suspension that step (5) is obtained is with athomin, Bravo, citric acid and nimbin according to 1:
0.1: 0.2: 0.01: 0.5 is well mixed, is drying to obtain described long-acting biological nematicidal composition.
Embodiment 3
A kind of long-acting biological kills the preparation method of nematode combination agent, including：
Step (1), by the Metarhizium anisopliae (Metarhizium guizhouense) of Cord blood, strain transfer is extremely
On PDA culture medium inclined-plane, cultivated 2-4 days under the conditions of 25-35 DEG C, realize the abundant activation of strain, the strain after activation culture connects
Kind shaking flask carries out seed culture；
Step (2), Shake flask medium is selected, consisting of the parts by weight of glucose 2, MgSO40.5 parts by weight, KCl 0.2
Parts by weight, ZnSO40.2 parts by weight, K2HPO40.6 parts by weight, the parts by weight of agar 2, K2HPO40.5 parts by weight and distilled water 30
Parts by weight；
Shaking flask is directly inoculated with using culture medium when shaking flask is inoculated with, the parameter of Shaking culture is as follows：Shaking speed is 150rpm,
Cultivation temperature is 29 DEG C, time 50h；Produce strain；
Step (3), prepares initial fermentation fluid nutrient medium, and the initial fermentation fluid nutrient medium includes：The weight of glucose 12
Measure part, the parts by weight of yeast 3, MgSO43 parts by weight, the parts by weight of KCl 1, ZnSO41 parts by weight, the parts by weight of distilled water 1000；
Seeding tank is sterilized and carries out initial fermentation, inoculum concentration and inoculation temperature are specially：121 DEG C of sterilizings 30min, now pH
Scope is 6.5；Zymotic fluid is inoculated with by 4% volume ratio of canned amount, and inoculation temperature is 30 DEG C, and fermentation time is 24 hours；Tentatively
Fermentation gained seed liquor is in thick, and spore content reaches 6 × 107/ more than ml；
Step (4), prepares depth fermentation broth, and the depth fermentation broth includes the weight of glucose 30
Part, the parts by weight of yeast 12, MgSO48 parts by weight, the parts by weight of KCl 6, ZnSO45 parts by weight, the parts by weight of agricultural crop straw 100 and
The parts by weight of distilled water 2000；
The production tank of depth fermentation sterilizes, inoculum concentration and inoculation temperature are specially：Nutrient solution connects through 121 DEG C of sterilizing 30min
Kind 4% volume ratio of amount, inoculation temperature are less than 30 DEG C；28 DEG C of fermentation temperature, fermentation period are 50 hours；
Liquid gives birth to spore content 5 × 10 in depth fermentation gained bacterium solution8/ more than ml, dry cell weight is more than 1.8%；
Step (5), the bacterium solution obtained by step (4) is stirred in fermentation tank, disperse mycelia and spore, add bacterium solution
The activated carbon of the weight of weight 20% and the polysorbate60 of bacterium solution weight 1%, it is standby to be prepared into bacteria suspension；
Step (6), the bacteria suspension that step (5) is obtained is with athomin, Bravo, citric acid and nimbin according to 1:
0.5: 0.4: 0.1: 0.8 is well mixed, is drying to obtain described long-acting biological nematicidal composition.
Embodiment 4
Above-described embodiment 1-3 long-acting biologicals prepared are killed into nematode combination agent, are placed on crop root use.
It is important to note that above-mentioned embodiment is only the preferable implementation of technical scheme
Example, it is impossible to technical scheme is caused to limit, it is any to conceive the product fallen within the scope of the appended claims, invade
Violate the patent right of the present invention.
Claims (7)
1. a kind of long-acting biological kills the preparation method of nematode combination agent, it is characterised in that including：
Step (1), by the Metarhizium anisopliae strain transfer of Cord blood to PDA culture medium inclined-plane, trained under the conditions of 25-35 DEG C
Support 2-4 days, realize the abundant activation of strain, the strain inoculation shaking flask after activation culture carries out seed culture；
Step (2), Shake flask medium is selected, consisting of glucose 1-5 parts by weight, MgSO40.1-0.6 parts by weight, KCl
0.1-0.3 parts by weight, ZnSO40.2-0.3 parts by weight, K2HPO40.1-0.9 parts by weight, agar 1-3 parts by weight, K2HPO4
0.1-0.9 parts by weight and distilled water 20-50 parts by weight；
Shaking flask is directly inoculated with using culture medium when shaking flask is inoculated with, the parameter of Shaking culture is as follows：Shaking speed is 150rpm, culture
Temperature is 29-30 DEG C, time 48-70h；Produce strain；
Step (3), prepares initial fermentation fluid nutrient medium, and the initial fermentation fluid nutrient medium includes：Glucose 10-15 weight
Part, yeast 1-6 parts by weight, MgSO41-5 parts by weight, KCl 0.2-2 parts by weight, ZnSO40.2-3 parts by weight and distilled water 200-
2000 parts by weight；
Seeding tank is sterilized and carries out initial fermentation, inoculation condition is specially：121 DEG C of culture medium sterilizing 30min, now pH scopes exist
6.0-6.5；Zymotic fluid is inoculated with by 4% volume ratio of canned amount, and inoculation temperature is less than 30 DEG C, and fermentation time is 18-24 hours；
Seed liquor is in thick obtained by initial fermentation, and spore content reaches 6 × 107/ more than ml；
Step (4), prepares depth fermentation broth, and the depth fermentation broth includes glucose 20-50 weight
Part, yeast 10-15 parts by weight, MgSO46-10 parts by weight, KCl 5-9 parts by weight, ZnSO43-7 parts by weight, agricultural crop straw
50-200 parts by weight and distilled water 500-3000 parts by weight；
The production tank sterilizing of depth fermentation, inoculation condition are specially：Culture medium presses canned amount through 121 DEG C of sterilizing 30min, zymotic fluid
4% volume ratio inoculation, inoculation temperature be less than 30 DEG C；28 ± 1 DEG C of fermentation temperature, fermentation period are 42-50 hours；
Liquid gives birth to spore content 5 × 10 in depth fermentation gained bacterium solution8/ more than ml；
Step (5), the bacterium solution obtained by step (4) is stirred in fermentation tank, disperse mycelia and spore, add bacterium solution weight
10-28% activated carbon and bacterium solution weight 0.01-2% polysorbate60, stirring, it is standby to be prepared into bacteria suspension；
Step (6), the bacteria suspension that step (5) is obtained is with athomin, Bravo, citric acid and nimbin according to 1-2: 0.1-
0.8: 0.2-0.5: 0.01-0.2: 0.5-1.2 is well mixed, and is drying to obtain described long-acting biological nematicidal composition.
2. a kind of long-acting biological according to claim 1 kills the preparation method of nematode combination agent, it is characterised in that step
(2) in, Shake flask medium composition is the parts by weight of glucose 2, MgSO40.5 parts by weight, the parts by weight of KCl 0.2, ZnSO40.2 weight
Measure part, K2HPO40.6 parts by weight, the parts by weight of agar 2, K2HPO40.5 parts by weight and the parts by weight of distilled water 30.
3. a kind of long-acting biological according to claim 1 kills the preparation method of nematode combination agent, it is characterised in that step
(3) in, the initial fermentation fluid nutrient medium includes：The parts by weight of glucose 12, the parts by weight of yeast 3, MgSO43 parts by weight, KCl
1 parts by weight, ZnSO41 parts by weight and the parts by weight of distilled water 1000.
4. a kind of long-acting biological according to claim 1 kills the preparation method of nematode combination agent, it is characterised in that step
(4) in, the depth fermentation broth includes the parts by weight of glucose 30, the parts by weight of yeast 12, MgSO48 parts by weight, KCl
6 parts by weight, ZnSO45 parts by weight, the parts by weight of agricultural crop straw 100 and the parts by weight of distilled water 2000.
5. a kind of long-acting biological according to claim 1 kills the preparation method of nematode combination agent, it is characterised in that step
(6) in, the bacteria suspension that step (4) obtains is with athomin, Bravo, citric acid and nimbin according to 1: 0.5: 0.4: 0.1:
0.8 is well mixed.
6. a kind of long-acting biological kills nematode combination agent, it is characterised in that is obtained by the preparation method of claim 1-5 any one
Arrive.
7. a kind of long-acting biological described in claim 6 kills the application of nematode combination agent, it is characterised in that is placed on root of the crop
Portion uses.
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| CN110169421A (en) * | 2019-05-20 | 2019-08-27 | 华中农业大学 | Application of the Metarhizium anisopliae in preparation prevention and treatment plant root-knot nematodes drug |
| CN111053087A (en) * | 2019-12-30 | 2020-04-24 | 江苏腾龙生物药业有限公司 | Horseradish pesticide and preparation method thereof |
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