CN108719208A - Entomopathogenic nematode large-scale method for producing and entomopathogenic nematode - Google Patents
Entomopathogenic nematode large-scale method for producing and entomopathogenic nematode Download PDFInfo
- Publication number
- CN108719208A CN108719208A CN201810461580.2A CN201810461580A CN108719208A CN 108719208 A CN108719208 A CN 108719208A CN 201810461580 A CN201810461580 A CN 201810461580A CN 108719208 A CN108719208 A CN 108719208A
- Authority
- CN
- China
- Prior art keywords
- nematode
- culture
- entomopathogenic
- wax moth
- greater wax
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 241000244206 Nematoda Species 0.000 title claims abstract description 202
- 230000000967 entomopathogenic effect Effects 0.000 title claims abstract description 63
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 55
- 241000255896 Galleria mellonella Species 0.000 claims abstract description 53
- 241000894006 Bacteria Species 0.000 claims abstract description 49
- 230000002538 fungal effect Effects 0.000 claims abstract description 39
- 239000001963 growth medium Substances 0.000 claims abstract description 36
- 238000009395 breeding Methods 0.000 claims abstract description 31
- 230000001488 breeding effect Effects 0.000 claims abstract description 31
- 239000000725 suspension Substances 0.000 claims abstract description 30
- 238000000926 separation method Methods 0.000 claims abstract description 20
- 238000004043 dyeing Methods 0.000 claims abstract description 16
- 238000000855 fermentation Methods 0.000 claims abstract description 16
- 230000004151 fermentation Effects 0.000 claims abstract description 16
- 210000000087 hemolymph Anatomy 0.000 claims abstract description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 27
- 239000000243 solution Substances 0.000 claims description 26
- 239000000843 powder Substances 0.000 claims description 18
- 241000238631 Hexapoda Species 0.000 claims description 16
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 16
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims description 10
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 10
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 9
- 229920002334 Spandex Polymers 0.000 claims description 9
- 240000008042 Zea mays Species 0.000 claims description 9
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 9
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 9
- 235000005822 corn Nutrition 0.000 claims description 9
- 235000013312 flour Nutrition 0.000 claims description 9
- 239000012530 fluid Substances 0.000 claims description 9
- 239000004759 spandex Substances 0.000 claims description 9
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 8
- 239000002285 corn oil Substances 0.000 claims description 8
- 235000005687 corn oil Nutrition 0.000 claims description 8
- 239000011780 sodium chloride Substances 0.000 claims description 8
- 238000003306 harvesting Methods 0.000 claims description 7
- 230000001954 sterilising effect Effects 0.000 claims description 7
- 238000004659 sterilization and disinfection Methods 0.000 claims description 7
- 230000031068 symbiosis, encompassing mutualism through parasitism Effects 0.000 claims description 6
- 241001480238 Steinernema Species 0.000 claims description 4
- 238000003113 dilution method Methods 0.000 claims description 4
- 238000004806 packaging method and process Methods 0.000 claims description 4
- 239000008280 blood Substances 0.000 claims description 3
- 210000004369 blood Anatomy 0.000 claims description 3
- 238000011049 filling Methods 0.000 claims description 3
- 238000000386 microscopy Methods 0.000 claims description 3
- 238000012856 packing Methods 0.000 claims description 3
- 206010042170 Strangury Diseases 0.000 claims description 2
- 238000011081 inoculation Methods 0.000 claims description 2
- 210000002751 lymph Anatomy 0.000 claims description 2
- 230000001717 pathogenic effect Effects 0.000 claims description 2
- 238000004321 preservation Methods 0.000 claims description 2
- 238000010923 batch production Methods 0.000 abstract description 3
- 238000000034 method Methods 0.000 description 16
- 239000000047 product Substances 0.000 description 13
- 239000007788 liquid Substances 0.000 description 7
- 230000000694 effects Effects 0.000 description 6
- 238000009630 liquid culture Methods 0.000 description 6
- 241000607479 Yersinia pestis Species 0.000 description 5
- 238000011031 large-scale manufacturing process Methods 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 238000012136 culture method Methods 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- 206010054949 Metaplasia Diseases 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000003670 easy-to-clean Effects 0.000 description 3
- 230000015689 metaplastic ossification Effects 0.000 description 3
- 230000007918 pathogenicity Effects 0.000 description 3
- 239000003053 toxin Substances 0.000 description 3
- 231100000765 toxin Toxicity 0.000 description 3
- 241000287828 Gallus gallus Species 0.000 description 2
- 241000500097 Heterorhabditis indica Species 0.000 description 2
- 241000509371 Steinernema feltiae Species 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 239000003124 biologic agent Substances 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 230000002349 favourable effect Effects 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 230000032696 parturition Effects 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000012372 quality testing Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000012549 training Methods 0.000 description 2
- 241000588921 Enterobacteriaceae Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241001523406 Heterorhabditis Species 0.000 description 1
- 208000000291 Nematode infections Diseases 0.000 description 1
- 235000019774 Rice Bran oil Nutrition 0.000 description 1
- 206010040047 Sepsis Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 230000000249 desinfective effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000000749 insecticidal effect Effects 0.000 description 1
- 239000002917 insecticide Substances 0.000 description 1
- 239000012774 insulation material Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000010977 jade Substances 0.000 description 1
- 238000009533 lab test Methods 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 239000008165 rice bran oil Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 208000013223 septicemia Diseases 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the production fields of entomopathogenic nematode, are related to entomopathogenic nematode large-scale method for producing and entomopathogenic nematode.The production method, including：Dip dyeing phase entomopathogenic nematode is inoculated on greater wax moth and is disseminated, after greater wax moth death, nematode is collected, nematode suspension is made；Greater wax moth is infected again using the nematode of separation, and then separation nematode carries out single bacterium culture out of dead greater wax moth polypide, and separation single bacterium culture nematode obtains expanding numerous nematode for producing；Greater wax moth live body is disseminated using phase entomopathogenic nematode is infected, then collects dead greater wax moth hemolymph, after the expansion of culture, identification, switching, fermentation and fungal component is numerous, obtains the fungal component bacterium solution of production；First obtained fungal component bacterium solution is accessed in the breeding bag for sharing culture medium, then obtained nematode is accessed in the breeding bag for sharing culture medium, culture.Easy to operate, good product quality of the invention, yield are higher with efficiency, it is easy to accomplish batch production mass propagation.
Description
Technical field
The invention belongs to the production technical fields of entomopathogenic nematode, are advised in particular to a kind of entomopathogenic nematode
Modelling production method and entomopathogenic nematode.
Background technology
Entomopathogenic nematode both at home and abroad studies it existing nearly 80 years history as a kind of biological control factor.This is not
Only it is if host range is extensive, to there is active search capability, there have to dwelling property of soil and borer pest to be special because it has many advantages
Preventive effect does not pollute the environment to safety of human and livestock, also not will produce resistance etc., and in many countries not by pollution regulations
Constraint it is easy to use, desinsection speed is fast, and can it is mixed with chemical insecticide and be widely used to prevention agricultural and herbage etc.
Pest.
Since this kind of nematode has the dual characteristics of natural enemy insect and pathogenic microorganism, can not only lead to as natural enemy insect
It crosses chemocepter actively to find and select host, and they carry symbiotic bacteria again, while infecting host's body cavity, with
Symbiotic bacteria is discharged, so that host is suffered from septicemia and dead, therefore this kind of nematode is referred to as entomopathogenic nematode.From 80 years 20th century
After generation, the Liquid Culture of entomopathogenic nematode has gradually moved towards scale and commercialized production road, merchandized handling
Company also constantly sets up.The basis of entomopathogenic nematode application be can inexpensive mass propgation, but currently used for nematode culture
Method be plate method, labour's consumption is big, unfavorable to the large-scale promotion application of nematode.Compared with solid culture, line
Worm Liquid Culture technology has labour's consumption low, but easily cause pollution, it is difficult to control when into large-scale production.
Therefore, a kind of solid-liquid two-phase eelworm culture method is researched and developed, solid culture technology and Liquid Culture technology are made
Have complementary advantages, maximize favourable factors and minimize unfavourable ones, it is both easy to operate, be easily achieved process manipulation, it is easy to clean, save culture space, ensure that
Product quality, the yield rate and cleannes of nematode improve, will be with wide commercial application prospect.
In consideration of it, special propose the present invention.
Invention content
The first object of the present invention is to provide a kind of entomopathogenic nematode large-scale method for producing, using two phase line of solid-liquid
Worm cultural method keeps solid culture technology and Liquid Culture technical advantage complementary, maximizes favourable factors and minimizes unfavourable ones, simple for process, easy to implement, side
Just it controls, good product quality, yield is higher with efficiency, it is easy to accomplish batch production mass propagation.
The second object of the present invention is that providing a kind of above-mentioned entomopathogenic nematode large-scale method for producing of utilization produces
The entomopathogenic nematode arrived.
To achieve the above object, the technical solution adopted by the present invention is：
According to an aspect of the present invention, the present invention provides a kind of entomopathogenic nematode large-scale method for producing, including such as
Lower step：
(a) dip dyeing phase entomopathogenic nematode is inoculated on greater wax moth live body, is disseminated at room temperature, waits for that greater wax moth is dead
After dying, nematode in water is collected, nematode suspension is made；Greater wax moth live body is infected again using the nematode suspension, then from dead
Separation nematode carries out single bacterium culture in the greater wax moth polypide died, and separation single bacterium culture nematode obtains for giving birth to after cultivating a period of time
Numerous nematode is expanded in production；
(b) using phase entomopathogenic nematode dip dyeing greater wax moth live body is infected, dead greater wax moth hemolymph is then collected, it will
Hemolymph is coated in media surface, and picking single bacterium colony obtains production after the expansion of identification, switching, fermentation and fungal component is numerous
Fungal component bacterium solution；
(c) the fungal component bacterium solution access first obtained step (b) shares in the breeding bag of culture medium, then step (a) is obtained
The nematode access arrived shares in the breeding bag of culture medium, and culture harvests nematode.
As further preferred technical solution, in step (a), dip dyeing phase entomopathogenic nematode is inoculated into greater wax moth live body
On, after cultivating 22~26h at 21~25 DEG C, after greater wax moth death, collects nematode in water within 6~8 days, nematode suspension is made
Liquid；
Preferably, separation nematode carries out single bacterium culture out of dead greater wax moth polypide, and 8~12 are cultivated at 21~25 DEG C
Single bacterium culture nematode is detached after it to obtain expanding numerous nematode for producing.
As further preferred technical solution, step (a) further includes：The nematode of separation is mixed with sponge, is then packed
With preservation, expand for production numerous.
As further preferred technical solution, nematode access that step (a) is obtained shares in the breeding bag of culture medium it
Before further include：
Sponge containing nematode is poured into funnel, so that the sponge containing nematode is laid on mesh screen, water is added not have sponge,
It squeezes sponge to empty water therein, after standing 0.5~2h, collects nematode suspension；
After the nematode suspension is carried out constant volume, nematode population is counted using dilution method step by step, it is outstanding to nematode after the completion of counting
Formalin solution disinfection is added in liquid, then again accesses nematode suspension in the breeding bag for sharing culture medium.
As further preferred technical solution, in step (b), lived using phase entomopathogenic nematode dip dyeing greater wax moth is infected
Body after cultivating 22~26h at 22~25 DEG C, collects dead greater wax moth hemolymph, and hemolymph is coated in culture base table by disinfection
Face is cultivated 3~5 days at 23~25 DEG C.
As further preferred technical solution, in step (b), the identification includes carrying out microscopy using microscope；Institute
The switching stated includes that fungal component is transferred on inclined-plane bacteria culture media, is cultivated 2~5 days at 23~25 DEG C；
Preferably, the fermentation include by the colony inoculation to the fermentation flask for filling zymotic fluid in slant tube,
23~25 DEG C, cultivate 2~4 days under 140~180rpm；
Preferably, the fungal component expand it is numerous include by culture after zymotic fluid carry out tap expand it is numerous, by one bottle of zymotic fluid
Expand numerous at most bottle or expand numerous to being bred in fermentation tank, ferments 2~4 days at 23~25 DEG C.
Further include that sponge is blended into 1~2cm in step (c) as further preferred technical solution3Fritter, be packed into
The step of breeding in bag, carrying out moist heat sterilization 1~2 hour；
Preferably, in step (c), the fungal component bacterium solution access for first obtaining step (b) shares in the breeding bag of culture medium,
It is cultivated 2~4 days at 23~25 DEG C, then the nematode access that step (a) is obtained shares in the breeding bag of culture medium, in 22~25
It is cultivated 10~12 days at DEG C, harvests nematode；
Preferably, the harvest nematode includes：Sponge containing nematode is poured into funnel, the sponge containing nematode is made
It is laid on mesh screen, water is added not have sponge, squeeze sponge and empty water therein, after standing 0.5~2h, collect nematode suspension；
After nematode suspension is centrifuged 2~5min at 2000~4000r/min, by nematode precipitate take out and with sterilizing sponge
Block mixes, and carries out finished product packing.
As further preferred technical solution, the shared culture medium includes：Sodium chloride, yeast, corn flour, egg powder,
Corn oil, bean powder, spandex sponge and water；
Preferably, the shared culture medium includes：25~30g of sodium chloride, 72~84g of yeast, 50~60g of corn flour, chicken
710~720g of powdered egg, 550~570g of corn oil, 1300~1320g of bean powder, 1510~1530g of spandex sponge and water 7100~
7200mL。
As further preferred technical solution, the entomopathogenic nematode is nematode or the heterorhabditis indica of genus steinernema
The nematode of category.
According to another aspect of the present invention, the entomopathogenic nematode scale metaplasia is utilized the present invention also provides a kind of
The entomopathogenic nematode that production method produces.
Compared with prior art, the beneficial effects of the present invention are：
The entomopathogenic nematode large-scale method for producing of the present invention first uses Liquid Culture mode culture to expand for producing respectively
When production expands numerous, large-scale production is carried out using the shared solid medium of nematode and fungal component for numerous nematode and fungal component, this
Sample, it is easy to operate, it is easy to accomplish process manipulates, easy to clean, can save culture space, it is also ensured that product quality, nematode
Yield, survival rate and cleannes improve, and dip dyeing pest has very strong pathogenicity, and cost of investment is relatively low, reduces production
Cost and technical difficulty have very real grind to further improving and promoting large-scale production entomopathogenic nematode technology
Study carefully and application value.
Specific implementation mode
Embodiment of the present invention is described in detail below in conjunction with embodiments and examples, but this field skill
Art personnel will be understood that following embodiments and embodiment are merely to illustrate the present invention, and be not construed as the model of the limitation present invention
It encloses.Based on the embodiments of the present invention, obtained by those of ordinary skill in the art without making creative efforts
Every other embodiment, shall fall within the protection scope of the present invention.The person that is not specified actual conditions, builds according to normal condition or manufacturer
The condition of view carries out.
In a first aspect, a kind of entomopathogenic nematode large-scale method for producing is provided at least one embodiment, including such as
Lower step：
(a) dip dyeing phase entomopathogenic nematode is inoculated on greater wax moth live body, is disseminated at room temperature, waits for that greater wax moth is dead
After dying, nematode in water is collected, nematode suspension is made；Greater wax moth live body is infected again using the nematode suspension, then from dead
Separation nematode carries out single bacterium culture in the greater wax moth polypide died, and separation single bacterium culture nematode obtains for giving birth to after cultivating a period of time
Numerous nematode is expanded in production；
(b) using phase entomopathogenic nematode dip dyeing greater wax moth live body is infected, dead greater wax moth hemolymph is then collected, it will
Hemolymph is coated in media surface, and picking single bacterium colony obtains production after the expansion of identification, switching, fermentation and fungal component is numerous
Fungal component bacterium solution；
(c) the fungal component bacterium solution access first obtained step (b) shares in the breeding bag of culture medium, then step (a) is obtained
The nematode access arrived shares in the breeding bag of culture medium, and culture harvests nematode.
Certain Pros and Cons are all individually present in existing entomopathogenic nematode plate method and liquid culture method；Separately
Outside, current living body propagation technology is mainly bred using greater wax moth, but is limited only to laboratory test breeding, yield mostly
It is extremely low, it can not batch production, large-scale production.In consideration of it, the present invention provides a kind of solid-liquid two-phase eelworm culture method, technology life
Production process is simple, and production efficiency is high, is easily manipulated, reduces production cost and technical difficulty, easy to clean, and it is empty to save culture
Between, it is ensured that product quality, yield and survival rate are high, and dip dyeing pest has very strong pathogenicity, and it is simple to breed equipment, drop
Low living body propagation cost.Live body cultural method has been effectively relieved in the production method of the present invention can not large-scale production Insect Pathogenic
The problem of nematode.
The method of the present invention is suitable for culture entomopathogenic nematode-genus steinernema (Steinernema) and heterorhabditis indica
Belong to (Heterorhabditis) nematode.It is particularly suitable for Steinernema feltiae industrial production method, including large-scale industrialized
Production method.The expansion for providing the kind of Steinernema feltiae is numerous, cultural method, the isolation and culture method of fungal component.The present invention
Belong to biological industry, both entomopathogenic nematode biological agent production method and production line, pest control is used for as biological agent.
It is understood that entomopathogenic nematode symbiotic bacteria is a kind of bacterium parasitized in entomopathogenic nematode enteron aisle,
Belong to enterobacteriaceae, amphimicrobian, chemoheterotrophy, Gram-negative.In nature, such bacterium is present in the enteron aisle of nematode
Interior, as nematode infects insect, fungal component is carried in insect bodies and is discharged into host's haemocoele, and fungal component is largely numerous
It grows, generates toxin and antibacterial substance, with nematode collective effect, kill host insect；In addition, fungal component can also decompose insect group
It knits, nutrition is provided for the growth and breeding of nematode and fungal component.
From the point of view of relationship of the entomopathogenic nematode with its fungal component, the insecticidal activity of nematode is mainly that fungal component is worked；
And fungal component is that after nematode infection host insect, fungal component is discharged into hemolymph using nematode as mediator, fungal component is in blood strangury
Palestine and China's mass propagation, generates toxin and nematode collective effect kills insect.The site of action of toxin is hemolymph, thus fungal component
There is higher injection activity.
In the present invention, dip dyeing phase entomopathogenic nematode is inoculated on greater wax moth live body, it is old to be preferably inoculated into greater wax moth
On ripe larva.Since entomopathogenic nematode has higher sensibility to greater wax moth, greater wax moth mature larva conduct is commonly used
What the recycling and acquisition of the host insect and nematode of living body propagation nematode detached traps insect.Entomopathogenic nematode is extensive
When producing metaplasia production, greater wax moth mature larva is that the raw of nematode product quality detection surveys insect.
Infection period (or dip dyeing phase) larva of entomopathogenic nematode is the worm age that can infect insect host；It is upper in application,
Infection period nematode has certain patience to environmental factor, and influential to insect.Therefore, the institute in common culture medium
The nematode of access is best to access infection period nematode.
In a preferred embodiment, above-mentioned entomopathogenic nematode large-scale method for producing includes：
(a) original seed nematode and expansion are numerous：It is covered in the culture dish of filter paper in bottom first, is put into greater wax moth mature larva, so
Be followed by into infecting phase nematode, it is typical but unrestricted after cultivating 22~26h at 21~25 DEG C, such as can be 21 DEG C, 22
DEG C, 23 DEG C, culture 22h at 24 DEG C or 25 DEG C, for 24 hours or after 26h, after greater wax moth death, be put in and collected on ware, 6~8 days
Afterwards, it such as can be to collect nematode in water after 6 days, 7 days or 8 days, it is spare that nematode suspension is made；
Infect greater wax moth mature larva again using the nematode of separation, later out of dead greater wax moth polypide detach nematode into
Row single bacterium culture, it is typical but unrestricted after being cultivated 8~12 days at 21~25 DEG C, such as can be 21 DEG C, 22 DEG C, 23
DEG C, cultivate 8 days, 9 days, 10 days, 11 days or 12 days at 24 DEG C or 25 DEG C after separation single bacterium culture nematode for produce expand it is numerous.
When production expands numerous, nematode based on bean powder, egg powder and fungal component is utilized to share culture medium progress scale metaplasia
Production.When elegans development to the phase of infecting (i.e. the 3rd instar larvae), nematode is detached out of culture medium, sampling carries out under anatomical lens
Quality testing counts every batch of by dilution method step by step later and breeds nematode population, then centrifuged to nematode suspension, by line
After worm precipitation is mixed with sponge, carries out product packaging and preserve.
(b) fungal component breeds：Greater wax moth mature larva is infected using phase entomopathogenic nematode is infected, is soaked at 22~25 DEG C
It is typical but unrestricted after contaminating 22~26h, such as can be that 22h, for 24 hours or 26h is infected at 22 DEG C, 23 DEG C, 24 DEG C or 25 DEG C
The disjunctive symbiosis bacterium out of dead greater wax moth larva body afterwards takes a drop death greater wax moth lymph to instill bacterium separation tablet training first
It supports on base, it is typical but unrestricted after being cultivated 3~5 days at 23~25 DEG C, such as can be at 23 DEG C, 24 DEG C or 25 DEG C
After culture 3 days, 4 days or 5 days, picking single bacterium colony is identified under the microscope, is then transferred in inclined-plane bacterial reproduction culture medium
On, it is cultivated 2~5 days at 23~25 DEG C, it is typical but unrestricted, such as can be to cultivate 2 at 23 DEG C, 24 DEG C or 25 DEG C
It, 3 days, 4 days or 5 days, it (such as can be triangle that the single bacterium colony in picking slant tube, which is inoculated into and fills the fermentation flask of zymotic fluid,
Bottle) in, it is cultivated 2~4 days at 23~25 DEG C, 140~180rpm, it is typical but unrestricted, such as can be at 23 DEG C, 24 DEG C
Or 25 DEG C, 140rpm, 150rpm, 160rpm or 180rpm shake culture is after 2 days, 3 days or 4 days, obtains original seed bacterium solution, then will
Production after original seed bacterium solution or expansion are numerous is accessed with fungal component bacterium solution to be shared containing nematode and fungal component in the breeding bag of culture medium,
It is cultivated 1~3 day at 23~25 DEG C, it is typical but unrestricted, such as can be to carry out Reproduction at 23 DEG C, 24 DEG C or 25 DEG C
1 day, 2 days or 3 days.
(c) expand jointly numerous：After fungal component bacterium solution culture culture 1~3 day, the original seed nematode that step (a) process is bred is connect
Enter to breed and continue to cultivate in bag, nematode is detached out of culture medium after 10~12 days, be carried out at the same time quality testing and count, determines every
Product packaging is carried out after the quality and quantity of batch and is preserved.
In a preferred embodiment, the shared culture medium includes：Sodium chloride, yeast, corn flour, egg powder,
Corn oil, bean powder, spandex sponge and water；
Preferably, the shared culture medium includes：25~30g of sodium chloride, 72~84g of yeast, 50~60g of corn flour, chicken
710~720g of powdered egg, 550~570g of corn oil, 1300~1320g of bean powder, 1510~1530g of spandex sponge and water 7100~
7200mL；
It is highly preferred that the shared culture medium includes：26~28g of sodium chloride, 75~80g of yeast, 54~58g of corn flour,
712~718g of egg powder, 555~565g of corn oil, 1305~1315g of bean powder, 1515~1525g of spandex sponge and water 7150~
7170mL；
Most preferably, the shared culture medium includes：Sodium chloride 28g, yeast 78g, corn flour 56g, egg powder 716g, jade
Rice bran oil 560g, bean powder 1310g, spandex sponge 1520g and water 7156mL.
The solid medium can steady production entomopathogenic nematode, reduce the risk that miscellaneous bacteria in incubation infects,
Effect stability has good effect of increasing production, is conducive to industrialized production entomopathogenic nematode.
Second aspect provides a kind of utilization above-mentioned entomopathogenic nematode large-scale method for producing at least one embodiment
Produce obtained entomopathogenic nematode.
With reference to specific embodiment, the invention will be further described.
Embodiment
A kind of entomopathogenic nematode large-scale method for producing, including：
1, the 1st day (infecting host insect)
Culture dish bottom is spread into one layer of filter paper, then greater wax moth mature larva is placed on culture dish filter paper, nematode is added
After suspension, culture dish is placed in 22~25 DEG C of insulating box and is cultivated for 24 hours.
2, the 2nd day (fungal component separation)
Greater wax moth hemolymph is collected after disinfecting the greater wax moth larva of infection in alcohol 5~10min, hemolymph is coated in training
Primary surface is supported, is cultivated 3~4 days under the conditions of being placed in 23~25 DEG C.
3, the 5th day or the 6th day (the symbiosis dientification of bacteria and switching)
After culture 3~4 days, picking single bacterium bacterium colony carries out microscopy (to be rod-shaped), transfers later under the microscope, in
Continue to cultivate on inclined-plane bacterial reproduction culture medium, equally be cultivated 3~4 days under the conditions of 23~25 DEG C.
4, the 8th day or the 9th day (fermentation)
The slant tube of culture 3 days or 4 days is taken out, bacterium colony is transferred in the fermentation flask for filling symbiosis bacteria culture fluid,
Be placed on shaking table and ferment 2 days at 23~25 DEG C, 150~160rpm.
5, the 10th day (fungal component is largely expanded numerous)
(1) fungal component is expanded numerous
The tap that zymotic fluid after culture carries out expands numerous, i.e., one bottle is expanded numerous at most bottle, is such as mass produced, using hair
The fermentation tank of fermentation tank such as 20L is bred, and will be connect flask (more bottles of zymotic fluids) and is placed in the shaking table top fermentation 2 under the conditions of 23~25 DEG C
It is to get to the symbiosis fermented liquid of production；
(2) breeding sponge prepares
Sponge is blended into 1cm3The fritter of left and right is packed into breeding bag, carries out moist heat sterilization about 1 hour.
6, the 12nd day (production)
(1) breeding bag prepares
Breeding bag can be carried out in the morning to prepare, that is, carry out culture medium in breeding bag and prepare, for example, can carry out according to the following formulation
Culture medium is prepared, and concrete operations are as follows：
Water 7156ml, NaCl28g, yeast 78g, corn flour 56g, egg powder 716g, corn oil 560g, bean powder 1310g and
Spandex sponge 1520g；
It sterilizes after prepared culture medium is packed into breeding bag, the condition of sterilizing for example can be to go out at 121 DEG C
Bacterium 30min.
(2) fungal component produces
After the symbiosis fermented liquid of obtained production is poured into breeding bag, breeding bag is put in hot room holder
On, it is cultivated 2 days at 23~25 DEG C；
7, the 14th day (nematode breeding)：
(1) nematode detaches
Sponge containing nematode is poured into big funnel, for example, the size of the big funnel can be 70cm × 70cm ×
40cm makes the sponge containing nematode be laid on mesh screen, and water is added and did not had sponge, squeezes sponge, and water in sponge is done net, quiet
After setting 1 hour, collecting nematode suspension (as produced for the first time, can first obtain in the way of described in above-mentioned steps (a) for producing
Expand numerous nematode, and nematode is precipitated and is mixed with sponge, then carry out nematode separation according still further to this method)；
(2) nematodal accounting
After nematode suspension is carried out constant volume, carries out dilution method step by step and count nematode population, after the completion of counting, to nematode suspension
Nematode suspension is connected to numerous by interior addition formalin after being sterilized 0.5~2 hour in 0.5%~1% formalin solution
(specific concentration can be depending on the specification of breeding bag, generally 20,000,000/bag) is grown in bag, is subsequently placed in hot room, 22
It is cultivated 10~12 days under the conditions of~25 DEG C.
8, the 26th day
(1) nematode separation (method was with the 14th day)
Sponge containing nematode is poured into big funnel, for example, the size of the big funnel can be 70cm × 70cm ×
40cm makes the sponge containing nematode be laid on mesh screen, and water is added and did not had sponge, squeezes sponge, and water in sponge is done net, quiet
After setting 1 hour, nematode suspension is collected；
(2) it cleans, pack
After the nematode suspension of finished product packing centrifuges 2~5min at 2000~4000r/min, nematode precipitation is taken out and gone out
Bacterium sponge block is mixed, and (matching criterion can be：50000000 are put into 50g sterilizing sponge blocks), then carry out finished product packet
Dress.
The technique effect that reaches of method of the present invention is：
(1) quality standard of finished product：
Regularity：By nematode suspension concentration dilution to 100-200IJs/ml, to the dip dyeing phase line of nematode under anatomical lens
The ratio of worm is detected, to reach the standard of 95% or so regularity；
Pathogenicity：Using greater wax moth mature larva as dip dyeing object, greater wax moth mature larva is infected at 23 ± 1 DEG C
The death rate of 24-72h, greater wax moth can reach 95.5% or more；
Survival rate：Finished product nematode is packaged as sponginum packaging, carries thermal insulation material, and the survival rate of nematode can reach 95%
More than.
(2) product storage and shelf-life：
Under conditions of 4-6 DEG C, the shelf-life is 6 months.
Finally it should be noted that：The above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Present invention has been described in detail with reference to the aforementioned embodiments for pipe, it will be understood by those of ordinary skill in the art that：Its according to
So can with technical scheme described in the above embodiments is modified, either to which part or all technical features into
Row equivalent replacement；And these modifications or replacements, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme.
Claims (10)
1. a kind of entomopathogenic nematode large-scale method for producing, which is characterized in that include the following steps：
(a) dip dyeing phase entomopathogenic nematode is inoculated on greater wax moth live body, is disseminated at room temperature, wait for greater wax moth death
Afterwards, nematode in water is collected, nematode suspension is made；Greater wax moth live body is infected again using the nematode suspension, then from death
Greater wax moth polypide in separation nematode carry out single bacterium culture, cultivate a period of time after separation single bacterium culture nematode obtain for producing
Expand numerous nematode；
(b) using phase entomopathogenic nematode dip dyeing greater wax moth live body is infected, dead greater wax moth hemolymph is then collected, by blood strangury
Bar be coated in media surface, picking single bacterium colony, by identification, switching, fermentation and fungal component expand it is numerous after, obtain the symbiosis of production
Bacterium bacterium solution；
(c) the fungal component bacterium solution access first obtained step (b) shares in the breeding bag of culture medium, then step (a) is obtained
Nematode access shares in the breeding bag of culture medium, and culture harvests nematode.
2. entomopathogenic nematode large-scale method for producing according to claim 1, which is characterized in that in step (a), will soak
Dye phase entomopathogenic nematode is inoculated on greater wax moth live body, after cultivating 22~26h at 21~25 DEG C, after greater wax moth death, and 6
It collects nematode in water within~8 days, nematode suspension is made；
Preferably, separation nematode carries out single bacterium culture out of dead greater wax moth polypide, after being cultivated 8~12 days at 21~25 DEG C
Separation single bacterium culture nematode obtains expanding numerous nematode for producing.
3. entomopathogenic nematode large-scale method for producing according to claim 1 or 2, which is characterized in that step (a) is also wrapped
It includes：The nematode of separation is mixed, then packaging and preservation with sponge, is expanded for production numerous.
4. entomopathogenic nematode large-scale method for producing according to claim 3, which is characterized in that obtain step (a)
Nematode access share culture medium breeding bag in before further include：
Sponge containing nematode is poured into funnel, the sponge containing nematode is made to be laid on mesh screen, water is added not have sponge, is squeezed
Sponge empties water therein, after standing 0.5~2h, collects nematode suspension；
After the nematode suspension is carried out constant volume, nematode population is counted using dilution method step by step, after the completion of counting, into nematode suspension
Formalin solution disinfection is added, then again accesses nematode suspension in the breeding bag for sharing culture medium.
5. entomopathogenic nematode large-scale method for producing according to claim 1, which is characterized in that in step (b), utilize
Phase entomopathogenic nematode dip dyeing greater wax moth live body is infected, after cultivating 22~26h at 22~25 DEG C, collects dead greater wax moth blood
Lymph, disinfection, is coated in media surface by hemolymph, is cultivated 3~5 days at 23~25 DEG C.
6. entomopathogenic nematode large-scale method for producing according to claim 1 or 5, which is characterized in that in step (b),
The identification includes carrying out microscopy using microscope；The switching includes that fungal component is transferred in inclined-plane bacteria culture media
On, it is cultivated 2~5 days at 23~25 DEG C；
Preferably, the fermentation include by the colony inoculation to the fermentation flask for filling zymotic fluid in slant tube, 23~
25 DEG C, cultivate 2~4 days under 140~180rpm；
Preferably, it includes that the zymotic fluid after culture is carried out tap to expand numerous that the fungal component, which is expanded numerous, one bottle of zymotic fluid is expanded numerous
At most bottle or expand numerous to being bred in fermentation tank, ferments 2~4 days at 23~25 DEG C.
7. entomopathogenic nematode large-scale method for producing according to claim 1, which is characterized in that in step (c), also wrap
It includes and sponge is blended into 1~2cm3Fritter, the step of being packed into breeding bag, carry out moist heat sterilization 1~2 hour；
Preferably, in step (c), the fungal component bacterium solution access for first obtaining step (b) shares in the breeding bag of culture medium, in 23
It is cultivated 2~4 days at~25 DEG C, then the nematode access that step (a) is obtained shares in the breeding bag of culture medium, at 22~25 DEG C
Culture 10~12 days harvests nematode；
Preferably, the harvest nematode includes：Sponge containing nematode is poured into funnel, the sponge containing nematode is made to tile
It on mesh screen, adding water not have sponge, squeezes sponge and empties water therein, after standing 0.5~2h, collect nematode suspension；
After nematode suspension is centrifuged 2~5min at 2000~4000r/min, nematode is precipitated and takes out and is mixed with sterilizing sponge block
It closes, carries out finished product packing.
8. entomopathogenic nematode large-scale method for producing according to claim 1, which is characterized in that the shared culture medium
Including：Sodium chloride, yeast, corn flour, egg powder, corn oil, bean powder, spandex sponge and water；
Preferably, the shared culture medium includes：25~30g of sodium chloride, 72~84g of yeast, 50~60g of corn flour, egg powder
710~720g, 550~570g of corn oil, 1300~1320g of bean powder, 1510~1530g of spandex sponge and water 7100~
7200mL。
9. entomopathogenic nematode large-scale method for producing according to claim 1, which is characterized in that the Insect Pathogenic line
Worm is the nematode of genus steinernema or the nematode of mass production.
10. the entomiasis produced using claim 1~9 any one of them entomopathogenic nematode large-scale method for producing
Former nematode.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810461580.2A CN108719208A (en) | 2018-05-15 | 2018-05-15 | Entomopathogenic nematode large-scale method for producing and entomopathogenic nematode |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810461580.2A CN108719208A (en) | 2018-05-15 | 2018-05-15 | Entomopathogenic nematode large-scale method for producing and entomopathogenic nematode |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108719208A true CN108719208A (en) | 2018-11-02 |
Family
ID=63937558
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810461580.2A Pending CN108719208A (en) | 2018-05-15 | 2018-05-15 | Entomopathogenic nematode large-scale method for producing and entomopathogenic nematode |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108719208A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109644945A (en) * | 2018-12-14 | 2019-04-19 | 广东省生物资源应用研究所 | A kind of method of fast quick-recovery Steinernema carpocapsae infection phase nematode |
| CN113615649A (en) * | 2021-09-24 | 2021-11-09 | 河南省农业科学院 | Purification method and application of new soybean cyst nematode microspecies X12 |
| CN114916504A (en) * | 2022-06-08 | 2022-08-19 | 扬州绿源生物化工有限公司 | Steelinia sinensis culture method, Steelinia sinensis pupa capsule preparation and preparation method thereof |
| CN116875499A (en) * | 2023-07-06 | 2023-10-13 | 吉林省农业科学院 | Entomopathogenic nematode symbiotic bacteria and application of metabolite thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6184434B1 (en) * | 1992-05-15 | 2001-02-06 | The United States Of America As Represented By The Secretary Of Agriculture | Steinernema sp. nematode for suppression of Helicoverpa zea and Spodoptera frugiperda |
| CN1928065A (en) * | 2006-01-24 | 2007-03-14 | 中国科学院东北地理与农业生态研究所 | Industrial high power breeding method for entomopathogenic nematode alive body |
| CN105802893A (en) * | 2016-05-09 | 2016-07-27 | 南京农业大学 | Entomopathogenic nematode symbiotic bacteria strain and application thereof |
| CN106035250A (en) * | 2016-08-18 | 2016-10-26 | 浙江绿神天敌生物技术有限公司 | Entomopathogenic nematode culture process |
| CN106070091A (en) * | 2016-08-18 | 2016-11-09 | 浙江绿神天敌生物技术有限公司 | A kind of entomopathogenic nematode one-level kind source preparation method |
| CN107027715A (en) * | 2017-05-11 | 2017-08-11 | 中国科学院东北地理与农业生态研究所 | A kind of entomopathogenic nematode living body propagation method |
-
2018
- 2018-05-15 CN CN201810461580.2A patent/CN108719208A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6184434B1 (en) * | 1992-05-15 | 2001-02-06 | The United States Of America As Represented By The Secretary Of Agriculture | Steinernema sp. nematode for suppression of Helicoverpa zea and Spodoptera frugiperda |
| CN1928065A (en) * | 2006-01-24 | 2007-03-14 | 中国科学院东北地理与农业生态研究所 | Industrial high power breeding method for entomopathogenic nematode alive body |
| CN105802893A (en) * | 2016-05-09 | 2016-07-27 | 南京农业大学 | Entomopathogenic nematode symbiotic bacteria strain and application thereof |
| CN106035250A (en) * | 2016-08-18 | 2016-10-26 | 浙江绿神天敌生物技术有限公司 | Entomopathogenic nematode culture process |
| CN106070091A (en) * | 2016-08-18 | 2016-11-09 | 浙江绿神天敌生物技术有限公司 | A kind of entomopathogenic nematode one-level kind source preparation method |
| CN107027715A (en) * | 2017-05-11 | 2017-08-11 | 中国科学院东北地理与农业生态研究所 | A kind of entomopathogenic nematode living body propagation method |
Non-Patent Citations (2)
| Title |
|---|
| 付俊瑞等: ""昆虫病原线虫异小杆属新种Heterorhabditis beicherriana共生菌的分离及致病性"", 《中国生物防治学报》 * |
| 喻子牛等: "《农业微生物研究及产业化进展》", 29 February 2004, 中国农业出版社 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109644945A (en) * | 2018-12-14 | 2019-04-19 | 广东省生物资源应用研究所 | A kind of method of fast quick-recovery Steinernema carpocapsae infection phase nematode |
| CN109644945B (en) * | 2018-12-14 | 2022-07-26 | 广东省生物资源应用研究所 | Method for rapidly recovering nematodes in Steinernema carpocapsae infection stage |
| CN113615649A (en) * | 2021-09-24 | 2021-11-09 | 河南省农业科学院 | Purification method and application of new soybean cyst nematode microspecies X12 |
| CN114916504A (en) * | 2022-06-08 | 2022-08-19 | 扬州绿源生物化工有限公司 | Steelinia sinensis culture method, Steelinia sinensis pupa capsule preparation and preparation method thereof |
| CN114916504B (en) * | 2022-06-08 | 2023-10-13 | 江苏里下河地区农业科学研究所 | Culture method of Style-Johnsonii, style-Johnsonii pupa capsule preparation and preparation method thereof |
| CN116875499A (en) * | 2023-07-06 | 2023-10-13 | 吉林省农业科学院 | Entomopathogenic nematode symbiotic bacteria and application of metabolite thereof |
| CN116875499B (en) * | 2023-07-06 | 2024-04-09 | 吉林省农业科学院 | Entomopathogenic nematode symbiotic bacteria and application of metabolite thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108719208A (en) | Entomopathogenic nematode large-scale method for producing and entomopathogenic nematode | |
| CN104322451B (en) | The artificial low altitude area raising method of a kind of host of Cordyceps sinensis insect bat moth | |
| CN102578425B (en) | Artificial diet for Lepidopterous young larvae, preparation method for artificial diet and application of artificial diet | |
| CN105176828B (en) | Beauveria bassiana XNBb-04 strain and culture method thereof | |
| CN101215527A (en) | Method for cultivating silkworm chrysalis Cordyceps sinensis | |
| CN104642392B (en) | A kind of fumosorosea bacterium conidium wettable powder and its application | |
| CN102643148B (en) | Special biological disease-resistant vermin-proof culture medium for flowers and preparation method thereof | |
| CN102972446A (en) | Preparation method of steinernema carpocapsae | |
| CN101654665B (en) | Method for preparing Bacillus subtilis | |
| CN101157893A (en) | Large scale insect pathogenic nematodes living high-power culturing method | |
| CN107586725B (en) | Cordyceps liquid culture medium and method for culturing cordyceps by using same | |
| CN105815277A (en) | Solid culture method for entomopathogenic nematodes | |
| CN107164241A (en) | A kind of muscardine solid medium and preparation method thereof | |
| CN104988098B (en) | One plant of prevention root rot of beets and the Bacillus strain for promoting Sugarbeet Growth | |
| CN105557303A (en) | Indoor precise cultivation method of pleurotus tuber regium | |
| CN106035250A (en) | Entomopathogenic nematode culture process | |
| CN101451108B (en) | Verticillium lecanii for preventing and controlling fly type pests and use thereof | |
| CN1928065A (en) | Industrial high power breeding method for entomopathogenic nematode alive body | |
| CN101831396A (en) | Process for preparing oxytetracycline single colony frozen bacteria | |
| WO2023103304A1 (en) | Accurate large-scale indoor breeding method for chilo suppressalis using rice seedlings | |
| CN1325630C (en) | Culture of nematoda without bacteria | |
| CN105505799A (en) | Fermentation method and application of trichoderma viride mutant strains | |
| CN106591153B (en) | One plant of Metarhizium Strains and its application to carpocapsa pononella highly pathogenicity | |
| CN113455468B (en) | Industrialized breeding method of sterile cordyceps sinensis host larvae | |
| CN109169534A (en) | Nematode solid medium and its preparation method and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20181102 |