CN101856015A - Method for refrigerated storage of heterorhabditis bacteriophora entomopathogenic nematodes in cold areas - Google Patents
Method for refrigerated storage of heterorhabditis bacteriophora entomopathogenic nematodes in cold areas Download PDFInfo
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- CN101856015A CN101856015A CN200910071751A CN200910071751A CN101856015A CN 101856015 A CN101856015 A CN 101856015A CN 200910071751 A CN200910071751 A CN 200910071751A CN 200910071751 A CN200910071751 A CN 200910071751A CN 101856015 A CN101856015 A CN 101856015A
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Abstract
The invention provides a method for the refrigerated storage of heterorhabditis bacteriophora entomopathogenic nematodes in cold areas, which relates to the technical field of biological refrigerated storage and solves the problems of short storage period, difficult long-distance transportation, low survival rate and low pathogenicity of the conventional low temperature (over 0 DEGC) storage of entomopathogenic nematodes. The method comprises the following steps of: 1) preparing storage media and storage bags; 2) inoculating; 3) performing refrigerated storage at different temperatures; 4) defreezing; and 5) isolating nematodes. The method for the refrigerated storage of the heterorhabditis bacteriophora entomopathogenic nematodes in the cold areas performs the refrigerated storage at the different temperatures on the heterorhabditis bacteriophora entomopathogenic nematodes which are peculiar to high-cold areas, breaks the limitations of current production scale and application areas of the entomopathogenic nematodes to ensure a storage period of 5 to 6 months, a rate of the recovering of the nematodes from the storage medium of 90 percent, a defrozen nematode survival rate of over 80 percent and a soil insect prevention and control effect of over 80 percent, solves the problems of unfavorable long-distance transportation and obviously decreased pathogenicity of the nematodes which are stored at the temperature of over 0 DEG C for long time and achieves a good effect of biologically preventing and controlling destructive insects in agricultural productive practice.
Description
Technical field
The present invention relates to biological stored frozen technical field, specifically a kind of Han Qu of being used for has a liking for the alternating temperature stored frozen method of the different little bar of bacterium (Heterorhabditis bacteriophora) entomopathogenic nematode.
Background technology
As everyone knows, entomopathogenic nematode (Entomopathogenic nematodes) is a kind of very ergastic biological control factor that grew up 20 beginnings of the century, be meant to carry in the body to have pathogenic symbiotic bacteria, can cause the class parasitic nematode that insect is caused a disease.Successfully be used for control of grubs, mushroom fly, little carpenter moth, lichee and intended kind of insect surplus carpenter moth, anoplophora glabripennis and the oranges and tangerines weevil etc. 250.Exactly because entomopathogenic nematode has wide spectrum, characteristics such as efficient and safe for human and animal and environment, more than 40 countries such as the U.S., Australia, Germany, New Zealand, Canada, Japan, Russia and Holland have dropped into great amount of manpower and financial resources, carry out aspect researchs such as a large amount of nematode basic theories, field application and product development, made the application of entomopathogenic nematode and propagation technique obtain develop rapidly.The storage of entomopathogenic nematode is the focus of present entomopathogenic nematode area research.Usually entomopathogenic nematode takes the low-temperature storage mode to preserve, generally 8-14 ℃ under, can deposit 2-4 month, short, long-distance transport difficulty of storage life, survival rate is low and pathogenicity a little less than, thereby limited the production scale and the application area of entomopathogenic nematode; Liquid nitrogen can be used for preserving entomopathogenic nematode, but stored prod cost height is inconvenient again in batches.Most commercial nematodes are not low temperature resistant at present, if fresh nematode directly is put into below 0 ℃, nematode can cause death cell rupture because of forming ice-nucleus in the somatic cell.
Summary of the invention
The objective of the invention is for solve that there is short, long-distance transport difficulty of storage life in traditional low-temperature storage entomopathogenic nematode more than 0 ℃, survival rate is low and pathogenicity a little less than problem, provide a kind of Han Qu of being used for to have a liking for the different little bar of bacterium and belong to entomopathogenic nematode stored frozen method.
It is as follows that a kind of Han Qu of being used for has a liking for the different little bar entomopathogenic nematode stored frozen method concrete operations step of bacterium:
Step 1, prepare storage medium and reservoir bag: storage medium is a vermiculite, and vermiculite is crossed the 40-60 mesh sieve, removes broken end, keeps air capacity of soils, 121 ℃ of moist heat sterilization 25-30min then, and adding water to the vermiculite water content is 45-60%; Reservoir bag is a 80-100 purpose nylon net bag, and size is 10 * 20cm, and storage medium is put into reservoir bag;
Step 2, inoculation: the entomopathogenic nematode (infective juvenile IJs) that the new breeding of 10 ℃ of preservations is come out is inoculated in the storage medium vermiculite; By 10
4-5The dose inoculation infective juvenile of IJs/g vermiculite;
Step 3, alternating temperature stored frozen: postvaccinal vermiculite is put into refrigerator, place 4-5 ℃ of environment 24h, the speed with 1 ℃/10min cools to-1 ℃ afterwards, and at-1 ℃ of constant temperature 3h, and then cool to-4--5 ℃ with the speed of 1 ℃/10min, and-4--5 ℃ preservation; Promptly finish the stored frozen of entomopathogenic nematode;
Step 4, thaw: the speed with 1 ℃/7min is warming up to 4 ℃, 4 ℃ stop 1h after, place the room temperature 24h that thaws again, standby;
The separation of step 5, nematode: the nylon net bag that vermiculite will be housed places 15-25 ℃ of water to soak, and behind the taking-up nylon net bag, soak is nematode suspension, can be used for control and uses.
Described entomopathogenic nematode be separate in the high and cold area soil, filter out have a liking for the different little bar entomopathogenic nematode of bacterium.
But described refrigerator is the refrigerator with the temperature adjustment of alternating temperature district, can realize that alternating temperature is freezing.
Technique effect: the inventive method has been broken current entomopathogenic nematode production scale and has been used the limitation of area, storage life reaches 5-6 month, the back nematode survival rate of thawing reaches more than 80%, the nematode rate of recovery reaches 90% in the storage medium, before the target pest pathogenicity was higher than low-temperature storage, control soil insect effect reached more than 80%.Solve the difficult problem that unfavorable long-distance transport and the long term storage more than 0 ℃ obviously descend the nematode pathogenicity, realized the good result of biological control of insect pests in the agricultural production practice.
Embodiment:
A kind of Han Qu of being used for has a liking for the different little bar entomopathogenic nematode stored frozen method of bacterium, after the phase nematode is infected in inoculation to storage medium, reservoir bag is placed refrigerator, and is freezing by alternating temperature, obtains the best stored frozen condition of entomopathogenic nematode.
Technical solution of the present invention is not limited to following cited embodiment, also comprises any combination between each embodiment.
Embodiment one: present embodiment is used for Han Qu, and to have a liking for the concrete operations step of the different little bar entomopathogenic nematode stored frozen method of bacterium as follows:
Step 1, prepare storage medium and reservoir bag: storage medium is a vermiculite, and vermiculite is crossed the 40-60 mesh sieve, removes broken end, keeps air capacity of soils, 121 ℃ of moist heat sterilization 25-30min then, and adding water to the vermiculite water content is 45-60%; Reservoir bag is a 80-100 purpose nylon net bag, and size is 10 * 20cm, and storage medium is put into reservoir bag;
Step 2, inoculation: the entomopathogenic nematode (infective juvenile IJs) that the new breeding of 10 ℃ of preservations is come out is inoculated in the storage medium vermiculite; By 10
4-5The dose inoculation infective juvenile of IJs/g vermiculite;
Step 3, alternating temperature stored frozen: postvaccinal vermiculite is put into refrigerator, place 4-5 ℃ of environment 24h, the speed with 1 ℃/10min cools to-1 ℃ afterwards, and at-1 ℃ of constant temperature 3h, and then cool to-4--5 ℃ with the speed of 1 ℃/10min, and-4--5 ℃ preservation; Promptly finish the stored frozen of entomopathogenic nematode;
Step 4, thaw: the speed with 1 ℃/7min is warming up to 4 ℃, 4 ℃ stop 1h after, place the room temperature 24h that thaws again, standby;
The separation of step 5, nematode: the nylon net bag that vermiculite will be housed places 15-25 ℃ of water to soak, and behind the taking-up nylon net bag, soak is nematode suspension, can be used for control and uses.
Embodiment two: what present embodiment and embodiment one were different is that vermiculite is crossed 50 mesh sieves in the step 1.Other step and parameter are identical with embodiment one.
Present embodiment
Embodiment three: what present embodiment and embodiment one were different is that nylon net bag is 80 orders in the step 1.Other step and parameter are identical with embodiment one.
Embodiment four: what present embodiment and embodiment one were different is that the vermiculite water content is 50% in the step 1.Other step and parameter are identical with embodiment one.
Embodiment five: that present embodiment and embodiment one are different is 121 ℃ of moist heat sterilization 30min in the step 1.Other step and parameter are identical with embodiment one.
Embodiment six: present embodiment and embodiment one are different be in the step 2 entomopathogenic nematode by 10
4The dose inoculation infective juvenile of IJs/g vermiculite.Other step and parameter are identical with embodiment one.
Embodiment seven: present embodiment and embodiment one are different is that the water temperature of soaking the nylon net bag that vermiculite is housed in the step 5 is 20 ℃.Other step and parameter are identical with embodiment one.
Embodiment eight: present embodiment is used for Han Qu, and to have a liking for the concrete operations step of the different little bar entomopathogenic nematode stored frozen method of bacterium as follows:
Step 1, prepare storage medium and reservoir bag: storage medium is a vermiculite, and vermiculite is crossed 50 mesh sieves, removes broken end, keeps air capacity of soils, 121 ℃ of moist heat sterilization 30min then, and adding water to the vermiculite water content is 50%; Reservoir bag is 80 purpose nylon net bags, and size is 10 * 20cm, and storage medium is put into reservoir bag;
Step 2, inoculation: the entomopathogenic nematode (infective juvenile IJs) that the new breeding of 10 ℃ of preservations is come out is inoculated in the storage medium vermiculite; By 10
4The dose inoculation infective juvenile of IJs/g vermiculite;
Step 3, alternating temperature stored frozen: postvaccinal vermiculite is put into refrigerator, place 4-5 ℃ of environment 24h, the speed with 1 ℃/10min cools to-1 ℃ afterwards, and at-1 ℃ of constant temperature 3h, and then cool to-4--5 ℃ with the speed of 1 ℃/10min, and-4--5 ℃ preservation; Promptly finish the stored frozen of entomopathogenic nematode;
Step 4, thaw: the speed with 1 ℃/7min is warming up to 4 ℃, 4 ℃ stop 1h after, place the room temperature 24h that thaws again, standby;
The separation of step 5, nematode: the nylon net bag that vermiculite will be housed places 20 ℃ of water to soak, and behind the taking-up nylon net bag, soak is nematode suspension, can be used for control and uses.
Year December in January, 2006-2007, the inventor is from high and cold area---and the highly pathogenicity that separates the Harbin City, Heilongjiang Province city sylvan life soil, screens is had a liking for the different little bar of bacterium (Heterorhabditis bacteriophora) entomopathogenic nematode, carried out stored frozen investigative test research by above embodiment year December in January, 2007 to 2008, obtained the success that China different little bar in the cold district of cold district highly pathogenicity belongs to entomopathogenic nematode alternating temperature stored frozen.Thaw back nematode survival rate more than 80%, to the fresh nematode that the pathogenicity of greater wax moth and soil insect is come out apparently higher than firm breeding, the nematode rate of recovery reaches 90% in the storage medium, and control soil insect effect reaches more than 80%, both obtain economic benefit, obtained ecological benefits again.
Having a liking for the different little bar entomopathogenic nematode of bacterium and all can adopt the method to carry out stored frozen in the high and cold area soil.
Han Qu of the present invention has a liking for the different little bar of bacterium (Heterorhabditis bacteriophora) entomopathogenic nematode and is widely used in controlling underground pest and fruit trees such as potted plant crop, economic crops, vegetables, herbage, lawn, views and admires disguised control of insect on the forests such as gardens.
Claims (7)
1. one kind is used for Han Qu and has a liking for the different little bar entomopathogenic nematode stored frozen method of bacterium, it is characterized in that the stored frozen method operates according to the following steps:
Step 1, prepare storage medium and reservoir bag: storage medium is a vermiculite, and vermiculite is crossed the 40-60 mesh sieve, removes broken end, 121 ℃ of moist heat sterilization 25-30min then, and adding water to the vermiculite water content is 45-60%; Reservoir bag is a 80-100 purpose nylon net bag, and storage medium is put into reservoir bag;
Step 2, inoculation: the entomopathogenic nematode that the new breeding of 10 ℃ of preservations is come out is inoculated in the storage medium vermiculite; By 10
4-5The dose inoculation infective juvenile of IJs/g vermiculite;
Step 3, alternating temperature stored frozen: postvaccinal vermiculite is put into refrigerator, place 4-5 ℃ of environment 24h, the speed with 1 ℃/10min cools to-1 ℃ afterwards, and at-1 ℃ of constant temperature 3h, and then cool to-4--5 ℃ with the speed of 1 ℃/10min, and-4--5 ℃ preservation; Promptly finish the stored frozen of entomopathogenic nematode;
Step 4, thaw: the speed with 1 ℃/7min is warming up to 4 ℃, 4 ℃ stop 1h after, place the room temperature 24h that thaws again;
The separation of step 5, nematode: the nylon net bag that vermiculite will be housed places 15-25 ℃ of water to soak, and behind the taking-up nylon net bag, soak is nematode suspension.
2. a kind of Han Qu of being used for according to claim 1 has a liking for the different little bar entomopathogenic nematode stored frozen method of bacterium, it is characterized in that vermiculite described in the step 1 crosses 50 mesh sieves.
3. a kind of Han Qu of being used for according to claim 1 has a liking for the different little bar entomopathogenic nematode stored frozen method of bacterium, it is characterized in that nylon net bag described in the step 1 is 80 orders.
4. a kind of Han Qu of being used for according to claim 1 has a liking for the different little bar entomopathogenic nematode stored frozen method of bacterium, it is characterized in that the vermiculite water content is 50% in the step 3.
5. a kind of Han Qu of being used for according to claim 1 has a liking for the different little bar entomopathogenic nematode stored frozen method of bacterium, it is characterized in that 121 ℃ of moist heat sterilization 30min described in the step 1.
6. a kind of Han Qu of being used for according to claim 1 has a liking for the different little bar entomopathogenic nematode stored frozen method of bacterium, it is characterized in that the different little bar in cold district described in the step 2 belongs to by 10
4The dose inoculation infective juvenile of IJs/g vermiculite.
7. a kind of Han Qu of being used for according to claim 1 has a liking for the different little bar entomopathogenic nematode stored frozen method of bacterium, and the water temperature that it is characterized in that in the step 5 soaking the nylon net bag that vermiculite is housed is 20 ℃.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102428909A (en) * | 2011-09-22 | 2012-05-02 | 中国林业科学研究院森林生态环境与保护研究所 | Cryopreservation and thawing method of Bursaphelenchus xyfolus |
| CN109452313A (en) * | 2018-12-07 | 2019-03-12 | 四川省农业科学院植物保护研究所 | A kind of entomopathogenic nematode preparation and preparation method and application |
| CN113243359A (en) * | 2021-05-07 | 2021-08-13 | 山东省农业科学院 | Aphid eating cecidomyiia refrigerating preservative and preservation method |
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| CN85101908A (en) * | 1985-04-01 | 1987-01-10 | 澳大利亚生物科技公司 | What nematode stored and transported improves one's methods |
| CN1335061A (en) * | 2000-07-20 | 2002-02-13 | 广东省昆虫研究所 | Storing method of entomopathogenic nematode |
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2009
- 2009-04-09 CN CN 200910071751 patent/CN101856015B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN85101908A (en) * | 1985-04-01 | 1987-01-10 | 澳大利亚生物科技公司 | What nematode stored and transported improves one's methods |
| CN1335061A (en) * | 2000-07-20 | 2002-02-13 | 广东省昆虫研究所 | Storing method of entomopathogenic nematode |
Non-Patent Citations (2)
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102428909A (en) * | 2011-09-22 | 2012-05-02 | 中国林业科学研究院森林生态环境与保护研究所 | Cryopreservation and thawing method of Bursaphelenchus xyfolus |
| CN109452313A (en) * | 2018-12-07 | 2019-03-12 | 四川省农业科学院植物保护研究所 | A kind of entomopathogenic nematode preparation and preparation method and application |
| CN109452313B (en) * | 2018-12-07 | 2020-09-22 | 四川省农业科学院植物保护研究所 | Entomopathogenic nematode preparation, preparation method and application |
| CN113243359A (en) * | 2021-05-07 | 2021-08-13 | 山东省农业科学院 | Aphid eating cecidomyiia refrigerating preservative and preservation method |
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