CN1335061A - Storing method of entomopathogenic nematode - Google Patents
Storing method of entomopathogenic nematode Download PDFInfo
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- CN1335061A CN1335061A CN 00117274 CN00117274A CN1335061A CN 1335061 A CN1335061 A CN 1335061A CN 00117274 CN00117274 CN 00117274 CN 00117274 A CN00117274 A CN 00117274A CN 1335061 A CN1335061 A CN 1335061A
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Abstract
The technological scheme of the present invention includes cleaning of entomopathogenic nematode, addition of filling material to prepare nematode liquid, granule or wettable powder, and placing CO2 adsorbent serving also as oxygen increasing agent and/or ammonia compound adsorbent in the entomopathogenic nematode storing environment. The CO2 adsorbent and ammonia compound adsorbent are packed independently in some package with ventilation holes and can adsorb CO2 and ammonia compound and supply oxygen essential for nematode to metabolize and survive. The present invention makes it possible to store entomopathogenic nematode for long period and transport it for long distance.
Description
The present invention relates to methods for bulk storage and transport of entomopathogenic nematodes as biopesticides.
The entomopathogenic Steinernematadae and Heterorhabditidae are novel biopesticides. Such nematodes enter the body of the insect in an infective stage in the form of an insect bite (IJ) with the host food or from the natural opening (e.g. anus, stomata) ofthe insect, the internode membrane, and subsequently release symbiotic bacteria of the genus Xenorhabdus (currently symbiotic with nematodes of the family siellaceae) or the genus photohabdus (currently symbiotic with nematodes of the family Xenorhabdus) carried in the intestinal lumen. The toxins (virulence factors) secreted by nematodes and commensal bacteria cause insect death. Entomopathogenic nematodes have a broad host range; the insect pest control agent has the capability of actively searching hosts, particularly soil-inhabiting and boring insects; is safe to human, livestock and environment. In recent years, the pesticide composition has been widely applied to control of pests such as agriculture, forestry, pasture and sanitation, is highly valued by scholars and commercial departments at home and abroad, and is commercialized.
The infective stage larvae of entomopathogenic nematodes are of an age to infest the insect host. Nematodes of this age do not feed and rely on stored energy substances in the body. They require oxygen and certain humidity conditions to survive for a longer period of time. The main limiting factor affecting the use of entomopathogenic nematodes as biopesticides is the storage and transport capacity of the nematodes during the infection phase. In the commercialization process of entomopathogenic nematode preparations, the effective shelf life of nematodes is prolonged, so that the optimal time selection for long-distance transportation and field application is facilitated, and the method is one of key technologies for nematode industrial production. The basic principle of nematode storage is that under certain environmental conditions, nematode metabolism is reduced, and the consumption in vivo is reduced to the lowest level capable of maintaining the life of the nematode and has certain tolerance to environmental stress; if necessary, the life activity can be restored to normal level under certain conditions.
Currently, there are two types of methods for storing nematodes: one is a preservation method in which the nematodes are dehydrated slowly at a certain humidity to enter a dry state (int. patent, WO/87/78515); in addition, the nematode is adsorbed and preserved by utilizing adsorptive filling materials such as sponge, diatomite, active carbon, polymer gel and the like. Currently, most patents in the world describe nematode storage by incorporating the nematode in the infective stage into various fillings. For example, Yukawa et al (U.S. patent4,765,275) describe a method of nematode storage and transport by mixing the nematodes with an adsorbent material, such as activated charcoal, and adding a bacteriostatic material. Pruitt&Friedman (U.S. Pat. No. 5,170,744) stores nematodes in gel packs with the addition of sucrose, glucose as stabilizers. Nelsen&Mannion (U.S. Pat. No. 4,615,883; 4,753,799) capsules the gels into nematodes. Bedding (U.S. patent5,042,427) uses various adsorptive soils to store nematodes. De Vlieger&Smits (U.S. Pat. No. 5,807,566) mixes decomposable polymers (such as proteins, lipopolysaccharides and the like) and ultraviolet light absorbable substances with nematodes to prepare dosage forms. Silver et al (int. patent, WO95/05077) prepares nematode capsules from various powdered substances (e.g., silica gel, clay, cellulose, starch, etc.) in combination with anti-mildew, bacteriostatic, emulsifying, stabilizing, and ultraviolet light absorbers. Chang&Gehret (U.S. patent5,401,506) also stores nematodes as various lipopolysaccharides, vegetable oils, insect baits and hydrogen peroxide (0.01-1.0% by weight). Although nematodes can be stored in various fillings, certain humidity and oxygen must be maintained. Nematodes are typically stored in relatively closed packages to facilitate moisture retention. However, the closed packaging environment affects the release of metabolic gases (e.g., carbon dioxide, ammonia-containing compounds, etc.) and the supply ofoxygen during nematode storage. In addition, elimination of metabolites and increased oxygen content in the storage during transport of nematodes, particularly during cryogenic transport of the nematode fluid, is important for nematode survival.
It is an object of the present invention to provide a method which can be used for the long-term storage and transport of entomopathogenic nematodes.
The invention provides a method for storing and transporting entomopathogenic nematodes for a long time, which adopts the technical scheme that: adding filling material such as water, diatomaceous earth, kaolin, various gels, cellulose or sponge into cleaned entomopathogenic nematode, making into liquid, granule or wettable powder, and storing in aerobic, anoxic or anaerobic environment. The method is characterized in that a carbon dioxide adsorbent or (and) a carbon dioxide adsorbent and an oxygen increasing agent are placed in a storage environment of entomopathogenic nematodes, the carbon dioxide adsorbent or (and) the carbon dioxide adsorbent and the oxygen increasing agent are packaged independently, and an air vent is reserved and placed in a nematode storage object.
The adsorbent for carbon dioxide according to the present invention may be any one of hydroxides, such as calcium hydroxide, sodium hydroxide, magnesium hydroxide, potassium hydroxide, etc., or a mixture of several kinds thereof. These hydroxides can adsorb carbon dioxide to form carbonates and water. Therefore, the concentration of carbon dioxide in the nematode dosage form can be reduced, and a good nematode storage environment can be maintained.
The carbon dioxide adsorbent and oxygen increasing agent can adopt any one of alkaline earth metal peroxides, such as calcium peroxide, sodium peroxide, potassium peroxide, magnesium peroxide and the like or a mixture ofa plurality of substances. The peroxides of these alkaline earth metals can absorb moisture in the nematode storage environment, forming hydroxides, which release oxygen. The hydroxide formed can adsorb carbon dioxide, and can reduce the concentration of carbon dioxide in the nematode dosage form.
The carbon dioxide adsorbent and oxygen increasing agent can also adopt a mixed package of hydroxide and hydrogen peroxide, wherein the hydrogen peroxide is separately packaged and is dropwise added into the hydroxide at a certain flow rate. The hydroxide, when mixed with hydrogen peroxide, releases oxygen, and the hydroxide itself adsorbs carbon dioxide. The reaction formula is as follows (taking the reaction of calcium hydroxide and hydrogen peroxide as an example):
the carbon dioxide adsorbent and oxygen increasing agent can also adopt a mixed package of alkaline earth metal peroxide and hydrogen peroxide, wherein the hydrogen peroxide is independently packaged and is dripped into the alkaline earth metal peroxide and the product thereof according to a certain flow rate. The peroxide can absorb moisture in the nematode storage environment, produce hydroxide and release oxygen. The hydroxide, when mixed with hydrogen peroxide, can evolve oxygen. The hydroxide formed can adsorb carbon dioxide. The reaction formula is as follows (taking calcium peroxide as an example):
in practice, peroxides are generally contained in ventable plastic bottles and release oxygen by absorbing moisture; hydroxides may also be contained in ventable plastic bottles; the plastic bottle filled with hydrogen peroxide can be placed in the ventable plastic bottle filled with peroxide or (and) hydroxide, and the hydrogen peroxide is dripped into the peroxide or (and) hydroxide according to a certain flow rate, and after the reaction, oxygen can be released. The mixed packaging bottle can not only release oxygen, but also absorb carbon dioxide.
A further measure of the invention is to place an ammonia-containing compound adsorbent in the storage environment of the entomopathogenic nematodes in order to exclude the ammonia-containing compound metabolites from the storage.
The ammonia-containing compound adsorbent can be selected from one or more of the following substances: natural Buddha stone and artificial Buddha stone. The Buddha stone is an aluminosilicate mineral containing alkali metal or alkaline earth metal, has unique adsorbability, ion exchange performance and larger adsorption surface, and can be used for reducing the content of ammonia and improving the storage environment of nematodes. When adding, it can be mixed with other adsorbent, or packaged independently, with vent left, and placed in nematode storage bag.
The entomopathogenic nematodes in the method of the invention mainly refer to nematodes in the infection phase. The invention is mainly suitable for entomopathogenic nematodes of species including Steinernemataceae (Steinernemanidae) and Heterorhabdidae (Heterorhabditidae). Preferred classes of entomopathogenic nematodes include Steinernema carpocapsae and Heterorhabditis bacteriophora.
When the method is used for storing the nematodes, the carbon dioxide adsorbent and the oxygen increasing agent and the ammonia-containing compound adsorbent are added, the adding amount of the carbon dioxide adsorbent, the carbon dioxide adsorbent and the oxygen increasing agent and the ammonia-containing compound adsorbent is not strictly limited, and the adding amount of the carbon dioxide adsorbent, the carbon dioxide adsorbent and the oxygen increasing agent and the ammonia-containing compound adsorbent are generally determined according to the type of the stored nematodes, the number of the nematodes, the.
The method of the invention is adopted to store the nematodes, and the carbon dioxide adsorbent and the oxygenating agent or (and) the ammonia-containing compound adsorbent are added to absorb the carbon dioxide and the ammonia-containing compound in the nematode storage environment and provide oxygen required by metabolism of the nematodes, thereby obviously improving the survival rate of the nematodes. The invention is an effective practical technical scheme for solving the problems of long-time storage and long-distance transportation of entomopathogenic nematodes. Is especially suitable for the closed package or liquid storage and transportation of the nematodes.
The following examples are further illustrative of the practice of the present invention and should not be construed as limiting thereof.
Example one
Entomopathogenic nematodes Steinernema carpocapsae a24 and heterhahabbacteroidiophora H06 were cultured as soy flour medium solids for 3 weeks, washed with tap water, and then concentrated. In a plastic bottle with a cap (diameter × height ═ 4.1 × 5.2cm), 5 g of cleaned nematodes (about 1 million nematodes) in the infection phase were charged, and 10 g of diatomaceous earth and 0.5 g of a mixture of natural phorbol were added. Then, 0.1 g of calcium hydroxide was placed in a small plastic vial (diameter. times. high. 1.4X 5cm), and then placed in a centrifuge tube (1.5ml) containing 0.2ml of hydrogen peroxide. A needle was used to drill a micro-hole in the bottom and top of the centrifuge tube to allow micro-drops of hydrogen peroxide to be added to the calcium hydroxide.The whole small plastic finger tube is placed in a plastic bottle, and the cover is screwed tightly. The control treatment did not add natural bergamot and calcium hydroxide and hydrogen peroxide. All treated plastic bottles were stored at 30 ℃ in a climatic chamber at a relative humidity of 80%. Samples were then taken periodically to check for nematode survival.
Results show that after 3,7 and 14 days of storage, the nematode survival rates of the Steinernema carpocapsae A24 treated plastic bottles (i.e. with the addition of natural bergamot, calcium hydroxide and hydrogen peroxide) were 98%, 86% and 61%, respectively; nematode survival rates in control plastic bottles were 63%, 7% and 0, respectively. Similarly, the nematode survival rates for the Heterorhabditis bacteriophora H06 nematodes after 3,7 and 14 days of storage were 96%, 94% and 77% for the treated plastic bottles (i.e., with the addition of natural bergamot, calcium hydroxide and hydrogen peroxide); nematode survival rates in control plastic bottles were 61%, 4% and 0, respectively. Therefore, after the natural Buddha stone, the calcium hydroxide and the hydrogen peroxide are added, the survival capability of the nematodes in high-temperature and closed packages can be obviously improved.
Example two
Entomopathogenic nematodes Steinernema carpocapsae a24 and heterhahabbacteroidiophora H06 were cultured as soy flour medium solids for 3 weeks, washed with tap water, and then concentrated. In a plastic bottle with a cap (diameter × height ═ 4.1 × 5.2cm), 5 g of cleaned nematodes (about 1 million nematodes) in the infection phase were charged, and 10 g of diatomaceous earth and 0.5 g of a mixture of natural phorbol were added. Then 0.1 g of calcium peroxide was placed in a small plastic vial (diameter. times. high. 1.4. times.5 cm). The whole small plastic finger tube is placed in a plastic bottle, and the cover is screwed tightly. The control treatment did not add natural bergamot and calcium peroxide. All treated plastic bottles were stored at 30 ℃ in a climatic chamber at a relative humidity of 80%. Samples were then taken periodically to check for nematode survival.
The results show that after 3,7 and 14 days of storage, the nematode survival rates of the Steinernema carpocapsae A24 treated plastic bottles (i.e. with the addition of natural stonite, calcium peroxide) were 91%, 76% and 39%, respectively; nematode survival rates in control plastic bottles were 63%, 7% and 0, respectively. Similarly, the nematode survival rates for the Heterorhabditis bacteriophora H06 nematodes after 3,7 and 14 days of storage were 86%, 74% and 41% for the treated plastic bottles (i.e., with the natural bergamot, calcium peroxide); nematode survival rates in control plastic bottles were 61%, 4% and 0, respectively. Therefore, after the natural Buddha stone and the calcium peroxide are added, the survival capability of the nematodes in high-temperature and closed packages can be obviously improved.
Claims (11)
1. A method for storing entomopathogenic nematodes, which comprises adding a filler to entomopathogenic nematodes to prepare a liquid, granules or wettable powder, and storing the mixture in an aerobic, anoxic or anaerobic environment, characterized in that a carbon dioxide adsorbent or (and) a carbon dioxide adsorbent and oxygenating agent is placed in the storage environment of entomopathogenic nematodes, and the carbon dioxide adsorbent or (and) the carbon dioxide adsorbent and oxygenating agent are packaged separately, leaving a vent and placed in a nematode storage.
2. The method of claim 1, wherein said carbon dioxide adsorbent is a hydroxide.
3. The method of claim 1, wherein said carbon dioxide adsorbent and oxygenating agent is an alkaline earth metal peroxide.
4. The method of claim 1, wherein the carbon dioxide adsorbent and oxygenating agent is a mixed package of hydroxide and hydrogen peroxide, and wherein the hydrogen peroxide is separately packaged and added dropwise to the hydroxide at a flow rate.
5. The method of claim 1, wherein the carbon dioxide adsorbent and oxygenating agent is a mixed package of alkaline earth metal peroxide and hydrogen peroxide, and the hydrogen peroxide is separately packaged and added dropwise to the alkaline earth metal peroxide and its resultant at a certain flow rate.
6. A method of storing entomopathogenic nematodes according to claim 2 or 4, wherein said hydroxide is one or a mixture of calcium hydroxide, sodium hydroxide, magnesium hydroxide and potassium hydroxide.
7. A method of storing entomopathogenic nematodes according to claim 3 or 5, wherein said alkaline earth metal peroxide is a mixture of one or more of calcium peroxide, sodium peroxide, potassium peroxide and magnesium peroxide.
8. The method of claim 1, wherein an ammonia-containing compound adsorbent is further disposed in the environment in which the entomopathogenic nematodes are stored.
9. The method for storing entomopathogenic nematodes according to claim 8, wherein said adsorbent containing ammonia compounds is natural Buddha stone or (and) artificial Buddha stone.
10. A method of storage of entomopathogenic nematodes according to claim 1, characterized in that said entomopathogenic nematodes comprise species of the families steinernemataceae (steinernematadae) and heterorhabdidae (heterorhabdidae).
11. The method of claim 10, wherein said entomopathogenic nematode species comprise Steinernema carpocapsae and Heterorhabditis bacteriophora.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB001172743A CN1184882C (en) | 2000-07-20 | 2000-07-20 | Storing method of entomopathogenic nematode |
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| CNB001172743A CN1184882C (en) | 2000-07-20 | 2000-07-20 | Storing method of entomopathogenic nematode |
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| CN1335061A true CN1335061A (en) | 2002-02-13 |
| CN1184882C CN1184882C (en) | 2005-01-19 |
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| CNB001172743A Expired - Fee Related CN1184882C (en) | 2000-07-20 | 2000-07-20 | Storing method of entomopathogenic nematode |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101856015A (en) * | 2009-04-09 | 2010-10-13 | 中国科学院东北地理与农业生态研究所 | Method for refrigerated storage of heterorhabditis bacteriophora entomopathogenic nematodes in cold areas |
| CN101305709B (en) * | 2008-06-13 | 2011-04-20 | 云南农业大学 | Application of micrometre flimsy in making the nematode permanent glassslide |
| CN106172259A (en) * | 2016-08-18 | 2016-12-07 | 浙江绿神天敌生物技术有限公司 | A kind of entomopathogenic nematode cleaning method |
| CN106962324A (en) * | 2017-04-28 | 2017-07-21 | 广东省生物资源应用研究所 | A kind of method of long-term storage entomopathogenic nematode |
| CN108142415A (en) * | 2017-12-26 | 2018-06-12 | 河北农业大学 | Method for preparing entomopathogenic nematode capsule |
| CN109845730A (en) * | 2019-04-03 | 2019-06-07 | 河北农业大学 | Powder containing entomopathogenic nematodes |
-
2000
- 2000-07-20 CN CNB001172743A patent/CN1184882C/en not_active Expired - Fee Related
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101305709B (en) * | 2008-06-13 | 2011-04-20 | 云南农业大学 | Application of micrometre flimsy in making the nematode permanent glassslide |
| CN101856015A (en) * | 2009-04-09 | 2010-10-13 | 中国科学院东北地理与农业生态研究所 | Method for refrigerated storage of heterorhabditis bacteriophora entomopathogenic nematodes in cold areas |
| CN101856015B (en) * | 2009-04-09 | 2013-01-16 | 中国科学院东北地理与农业生态研究所 | Method for refrigerated storage of heterorhabditis bacteriophora entomopathogenic nematodes in cold areas |
| CN106172259A (en) * | 2016-08-18 | 2016-12-07 | 浙江绿神天敌生物技术有限公司 | A kind of entomopathogenic nematode cleaning method |
| CN106962324A (en) * | 2017-04-28 | 2017-07-21 | 广东省生物资源应用研究所 | A kind of method of long-term storage entomopathogenic nematode |
| CN108142415A (en) * | 2017-12-26 | 2018-06-12 | 河北农业大学 | Method for preparing entomopathogenic nematode capsule |
| CN108142415B (en) * | 2017-12-26 | 2020-09-04 | 河北农业大学 | Method for preparing entomopathogenic nematode capsule |
| CN109845730A (en) * | 2019-04-03 | 2019-06-07 | 河北农业大学 | Powder containing entomopathogenic nematodes |
| CN109845730B (en) * | 2019-04-03 | 2022-02-11 | 河北农业大学 | Powder containing entomopathogenic nematodes |
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| CN1184882C (en) | 2005-01-19 |
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Granted publication date: 20050119 Termination date: 20100720 |