CN109644945B - Method for rapidly recovering nematodes in Steinernema carpocapsae infection stage - Google Patents
Method for rapidly recovering nematodes in Steinernema carpocapsae infection stage Download PDFInfo
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- CN109644945B CN109644945B CN201811533383.3A CN201811533383A CN109644945B CN 109644945 B CN109644945 B CN 109644945B CN 201811533383 A CN201811533383 A CN 201811533383A CN 109644945 B CN109644945 B CN 109644945B
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- 241000244206 Nematoda Species 0.000 title claims abstract description 29
- 238000000034 method Methods 0.000 title claims abstract description 21
- 241001480223 Steinernema carpocapsae Species 0.000 title claims description 4
- 208000015181 infectious disease Diseases 0.000 title abstract description 17
- 239000007788 liquid Substances 0.000 claims abstract description 22
- 210000000087 hemolymph Anatomy 0.000 claims abstract description 20
- 241000382353 Pupa Species 0.000 claims abstract description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 14
- 235000013601 eggs Nutrition 0.000 claims abstract description 13
- 239000012266 salt solution Substances 0.000 claims abstract description 11
- 102000002322 Egg Proteins Human genes 0.000 claims abstract description 8
- 108010000912 Egg Proteins Proteins 0.000 claims abstract description 8
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims abstract description 7
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims abstract description 7
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims abstract description 7
- 210000002969 egg yolk Anatomy 0.000 claims abstract description 7
- 235000013345 egg yolk Nutrition 0.000 claims abstract description 7
- 239000000843 powder Substances 0.000 claims abstract description 7
- 235000020183 skimmed milk Nutrition 0.000 claims abstract description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 6
- 239000008223 sterile water Substances 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 3
- 238000009472 formulation Methods 0.000 claims description 2
- 238000011084 recovery Methods 0.000 abstract description 17
- 230000000967 entomopathogenic effect Effects 0.000 abstract description 6
- 230000001524 infective effect Effects 0.000 abstract description 3
- 241000238631 Hexapoda Species 0.000 description 8
- 241000894006 Bacteria Species 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 238000011534 incubation Methods 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 4
- 241000607479 Yersinia pestis Species 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 241000255789 Bombyx mori Species 0.000 description 2
- 230000000749 insecticidal effect Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 235000006732 Torreya nucifera Nutrition 0.000 description 1
- 244000111306 Torreya nucifera Species 0.000 description 1
- 238000003287 bathing Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000853 biopesticidal effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 239000004973 liquid crystal related substance Substances 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 235000008935 nutritious Nutrition 0.000 description 1
- 210000004681 ovum Anatomy 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
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- 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
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- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention relates to a fast recoverySteinernema carpocapsaeThe invention relates to a method for nematode in infection phase, belonging to the technical field of entomopathogenic nematode cultureS.carpocapsaeInoculating the larvae in the infection stage into artificial egg liquid, recovering the larvae in the infection stage, and collecting recovered nematodes; the artificial egg liquid contains tussah pupa hemolymph, trehalose, egg yolk, skimmed milk powder, N's salt solution and water. By the process of the inventionS.carpocapsaeThe larvae in the infection stage can be quickly recovered, the proportion is high, the recovery rate after 2 days can reach 85.24 percent, and the method can be applied to quickly and efficiently recoverS.carpocapsaeLarvae in the infective stage, thereby being cultured efficiently and rapidlyS.carpocapsaeThe nematode provides conditions.
Description
Technical Field
The invention belongs to the technical field of entomopathogenic nematode culture, and particularly relates to rapid recoveryS. carpocapsaeA method for treating nematode in the infection stage.
Background
Entomopathogenic SphaeriaSteinernemaHeterorhamusHeterorhabditisNematodes are internationally novel high-potency biopesticides (Georgis)et al.2006) used for pest control since the 30 s of the twentieth century (Smart, 1995), has advantages of strong insecticidal ability, broad insecticidal spectrum, ability to actively search for hosts, safety to humans, animals and the environment, etc., is easy to mass-culture and formulate products, can be mixed with various chemical pesticides, and has special effects on boring and underground pests (Kaya and Gaugler 1993; grewalet al.，2005；Georgis et al.，2006；Yan et al.2013; pigment 29667, etc., 2014).
The life history of entomopathogenic nematodes begins with the introduction of symbiotic bacteria carried by the infected larvae (IJ) into the host insect, which evade the immune response of the insect and release the symbiotic bacteria in the insect hemolymph (downs and pets, 2002). The symbiotic bacteria proliferate in the hemolymph of the insect to kill the host insect, and the nematode feeds on the symbiotic bacteria to reproduce. When the insect is nutritionally depleted, 3-instar tolerant infected larvae form, are sufficiently moist in the external environment and warm enough, and leave the host insect carrying commensal bacteria (Brown and Gaugler, 1997) searching for new hosts. The infested larvae are the only free-living instar of the life history of entomopathogenic nematodes and can be applied using conventional sprayers to control pests. The larvae in the infection stage can survive for 1 to more months, and can be cultured industrially (Wright)et al.，2005）。
Recovery of infected larvae is critical in the liquid culture process. If the recovery rate is low, the number of first-generation adults is small, only a small portion of commensal bacteria is fed, and the offspring of the first-generation adults will form adults because they are sufficiently nutritious and will not develop into infected larvae. Because the quality of symbiotic bacteria in the culture solution is poor at this time, the second generation adults are difficult to generate offspring, so the yield of larvae in the infection stage in the culture process with the two generations of adults is lower than that in the culture process with only one generation of adults (Hirao and Ehlers, 2010), the culture time is long, and the shelf life of the final nematode product is shortened due to the fact that the culture solution contains a high proportion of nematodes in other ages at the time of harvesting.
Disclosure of Invention
To overcome the problems of the background art, the present invention provides a fast recoveryS. carpocapsaeThe method for the nematode in the infection phase has high recovery rate and is efficient and rapid in cultureS. carpocapsaeThe nematode provides conditions.
In order to realize the purpose, the invention is realized by the following technical scheme:
the block isRapid recoverySteinernema carpocapsaeThe method for treating the nematode in the infection stage specifically comprises the following steps:
1) blending and uniformly mixing tussah pupa hemolymph, trehalose, egg yolk, skimmed milk powder, N's salt solution and water to obtain artificial egg liquid;
2) will be provided withS. carpocapsaeAnd (4) inoculating the larvae in the infection stage into the artificial egg liquid, and collecting recovered nematodes after the larvae in the infection stage recover.
Preferably, the artificial egg liquid is prepared by the following method every 9 ml: 3 ml of tussah pupa hemolymph, 0.1 g of anhydrous trehalose, 2.5 ml of egg yolk, 0.1 g of skimmed milk powder, 1 ml of N's salt solution and 2.5 ml of sterile water are mixed uniformly.
Preferably, the formulation per liter of N's salt solution is as follows: NaCl 7.5 g/l, KCl 0.1 g/l, CaCl 2 0.2 g/l, NaHCO 3 0.2 g/liter, and the balance of water.
As a preference, the first and second liquid crystal compositions are,S. carpocapsaethe larvae in the infection stage areS. carpocapsae All infected stage larvae.
The recovered infected larvae can be inoculated into liquid or solid culture medium for further culture, thereby obtaining commercialized larvaeS. carpocapsaeLarvae in the infected stage.
The invention has the beneficial effects that:
S. carpocapsaethe larvae in the infection stage can recover in the artificial egg liquid quickly, the proportion is high, the recovery rate after 2 days reaches 85.24 percent, and the method can be applied to quickly and efficiently recoverS. carpocapsaeLarvae in the infective stage, thereby being cultured efficiently and rapidlyS. carpocapsaeThe nematode provides conditions.
Drawings
FIG. 1 is a graph comparing recovery of nematodes in example 1 and comparative examples 1-2.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, preferred embodiments of the present invention will be described in detail below to facilitate understanding of the skilled person.
Example 1
1) Entomopathogenic nematodes：S. carpocapsaeCollecting larvae (Infective juveniles) in an infected stage from dead bodies of the greater wax borers, filtering to remove dead insects, adjusting the concentration of nematodes to be 1000 IJ/ml, and storing at 15 ℃ and 100 rpm for later use. The nematode concentration was concentrated to 20000 IJ/ml at room temperature around 25 ℃ before the measurement.
2) Preparing artificial egg liquid: mixing 3 ml of tussah pupa hemolymph, 0.1 g of anhydrous trehalose, 2.5 ml of egg yolk, 0.1 g of skimmed milk powder, 1 ml of N's salt solution and 2.5 ml of sterile water uniformly, and storing at-20 deg.C (N's salt solution: NaCl 7.5 g, KCl 0.1 g, CaCl) 2 0.2 g，NaHCO 3 0.2 g of the suspension was added to 1000 ml of H 2 In O, 121 ℃, 10 5 Sterilizing under kPa for 30 min, and storing at room temperature for later use);
3) a4. mu.l of the incubation solution and 2. mu.l of the nematode solution (containing about 40 infection-stage nematodes) at a concentration of 20000 IJ/ml were added to each well of the 384-well plate to conduct the recovery culture.
Comparative example 1
The procedure was the same as in example 1, except that the incubation liquid was tussah pupa hemolymph.
Preparing tussah pupa hemolymph: cutting cocoons, taking out tussah pupae, bathing in water at 65 ℃ for 10 min, smearing pupae surface with 75% alcohol for disinfection, cutting pupae tail in a super-clean workbench, extruding and collecting hemolymph, and storing at-20 ℃ for later use;
comparative example 2
The procedure is the same as in example 1, except that the incubation liquid is tussah pupa hemolymph water mixed liquid.
Preparing a tussah pupa hemolymph water mixed solution: 3 ml of tussah pupa haemolymph and 6 ml of sterile water are mixed uniformly and are used in the spot.
Comparative example 3
The procedure was the same as in example 1 except that the incubation solution was used as a base solution.
Preparing a base liquid: mixing anhydrous trehalose 0.1 g, egg yolk 2.5 ml, skimmed milk powder 0.1 g, N's salt solution 1 ml and sterile water 5.5 ml, and storing at-20 deg.C (N's salt solution: NaCl 7.5 g, KCl 0.1 g, CaCl) 2 0.2 g，NaHCO 3 0.2 g was added to 1000 ml H 2 In O, 121 ℃，10 5 Autoclaving for 30 min under kPa, and storing at room temperature for use).
Analysis of experiments
Samples were taken at 6 wells per sampling time, daily, for 2 consecutive days, and 42 wells per nematode were treated. And adding water into the counting plate, sucking the nematodes in each hole into each hole of the counting plate, performing microscopic examination, and recording the number of the recovered nematodes in each hole.
S. carpocapsae The recovery rate of All was 0 after seven days of incubation in the basal fluid.
S. carpocapsaeThe recovery conditions of the infected larva in 3 kinds of incubation liquids (artificial ovum liquid, tussah pupa hemolymph, and tussah pupa hemolymph mixed liquid) are shown in figure 1,S. carpocapsaethe larvae in the infection stage recover fastest in the artificial egg liquid, the ratio is highest, the recovery rate after 2 d reaches 85.24 percent, the recovery rate is slightly higher than that in the tussah silkworm pupa hemolymph, and the recovery rate in the tussah silkworm pupa hemolymph mixed liquid is lowest, and is only 68.8 percent.
The method only needs a small amount of tussah pupa hemolymph to be matched with other substances, the recovery effect achieved by the method can be the same as that of the pure tussah pupa hemolymph, and tests prove that the artificial egg liquid is stored for one month at the temperature of 20 ℃ below zero, and the effect is as good as that of the just prepared artificial egg liquid.
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, while the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.
Claims (1)
1. A method for rapidly recovering nematodes in the infected phase of Steinernema carpocapsae, which is characterized by comprising the following steps: the method specifically comprises the following steps:
1) blending and uniformly mixing tussah pupa hemolymph, trehalose, egg yolk, skimmed milk powder, N's salt solution and water to obtain artificial egg liquid; every 9 ml of the artificial egg liquid is prepared by the following method: 3 ml of tussah pupa hemolymph, 0.1 g of anhydrous trehalose, 2.5 ml of egg yolk, 0.1 g of skimmed milk powder, 1 ml of N's salt solution and 2.5 ml of sterile water are uniformly mixed; the formulation of N's salt solution per liter is as follows: NaCl 7.5 g/L, KCl 0.1 g/L, CaCl 20.2 g/L, NaHCO 30.2 g/L, and water in balance;
2) and (3) inoculating the larvae in the infected stage of the carpocapsae All into the artificial egg liquid, and collecting recovered nematodes after the larvae in the infected stage recover.
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|---|---|---|---|---|
| CN1300817A (en) * | 1999-12-17 | 2001-06-27 | 广东省昆虫研究所 | Liquid culture method for pathogenic nematode of insect |
| CN101041810A (en) * | 2006-10-17 | 2007-09-26 | 广东省昆虫研究所 | Bacteria strain (KG strain) for culturing bait nematode and nematode culture method |
| CN103859210A (en) * | 2014-02-20 | 2014-06-18 | 广东省昆虫研究所 | Artificial medium of trichogramma |
| CN105831019A (en) * | 2016-04-21 | 2016-08-10 | 广东省昆虫研究所 | Method for quickly obtaining larvae of Steinernema carpocapsae at tidy age infection stage |
| CN108719208A (en) * | 2018-05-15 | 2018-11-02 | 吉林省怡科农业生物科技有限公司 | Entomopathogenic nematode large-scale method for producing and entomopathogenic nematode |
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| WO1994015460A1 (en) * | 1993-01-13 | 1994-07-21 | The University Of Florida | Biological control of orthoptera pest insects |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1300817A (en) * | 1999-12-17 | 2001-06-27 | 广东省昆虫研究所 | Liquid culture method for pathogenic nematode of insect |
| CN101041810A (en) * | 2006-10-17 | 2007-09-26 | 广东省昆虫研究所 | Bacteria strain (KG strain) for culturing bait nematode and nematode culture method |
| CN103859210A (en) * | 2014-02-20 | 2014-06-18 | 广东省昆虫研究所 | Artificial medium of trichogramma |
| CN105831019A (en) * | 2016-04-21 | 2016-08-10 | 广东省昆虫研究所 | Method for quickly obtaining larvae of Steinernema carpocapsae at tidy age infection stage |
| CN108719208A (en) * | 2018-05-15 | 2018-11-02 | 吉林省怡科农业生物科技有限公司 | Entomopathogenic nematode large-scale method for producing and entomopathogenic nematode |
Non-Patent Citations (1)
| Title |
|---|
| 生物杀虫剂-昆虫病原线虫的培养技术;颜珣等;《环境昆虫学报》;20160930(第5期);第1044-1051页 * |
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