CN109169534A - Nematode solid medium and its preparation method and application - Google Patents
Nematode solid medium and its preparation method and application Download PDFInfo
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- CN109169534A CN109169534A CN201811194454.1A CN201811194454A CN109169534A CN 109169534 A CN109169534 A CN 109169534A CN 201811194454 A CN201811194454 A CN 201811194454A CN 109169534 A CN109169534 A CN 109169534A
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- 241000244206 Nematoda Species 0.000 title claims abstract description 137
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- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 241000254109 Tenebrio molitor Species 0.000 claims abstract description 50
- 239000000843 powder Substances 0.000 claims abstract description 44
- 239000001963 growth medium Substances 0.000 claims abstract description 36
- 239000002609 medium Substances 0.000 claims abstract description 25
- 238000004108 freeze drying Methods 0.000 claims abstract description 20
- 239000007640 basal medium Substances 0.000 claims abstract description 19
- 230000000967 entomopathogenic effect Effects 0.000 claims abstract description 19
- 150000002632 lipids Chemical class 0.000 claims abstract description 9
- 235000013336 milk Nutrition 0.000 claims abstract description 4
- 210000004080 milk Anatomy 0.000 claims abstract description 4
- 239000008267 milk Substances 0.000 claims abstract description 4
- 239000000463 material Substances 0.000 claims abstract description 3
- 102000002322 Egg Proteins Human genes 0.000 claims description 16
- 108010000912 Egg Proteins Proteins 0.000 claims description 16
- 241000894006 Bacteria Species 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 14
- 210000002969 egg yolk Anatomy 0.000 claims description 12
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 4
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- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 241001465746 Heterorhabditidae Species 0.000 description 1
- 241000243785 Meloidogyne javanica Species 0.000 description 1
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- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 description 1
- 229910052794 bromium Inorganic materials 0.000 description 1
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- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
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- 235000019198 oils Nutrition 0.000 description 1
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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 present invention relates to nematode in-vitro solid production fields, and in particular to a kind of nematode solid medium and its preparation method and application.The nematode solid medium includes following components: (1) the Yellow meal worm larva powder of the freeze-drying of basal medium and (2) 1wt%~5wt%, the wherein basal medium serum-free, and do not include the lipid materials such as pluck and milk powder in the nematode solid medium.Nematode solid medium provided by the invention is remarkably improved the yield and quality of entomopathogenic nematode, while can be improved the efficiency of solids manufacture entomopathogenic nematode, saves the production time, reduces production cost.The death that prodenia litura can be dramatically speeded up using the nematode that culture medium of the invention produces, enhances the insecticidal power of nematode.
Description
Technical field
The present invention relates to nematode in-vitro solid production technical field more particularly to a kind of nematode solid medium and its preparations
Methods and applications.
Background technique
With the development of agricultural, crops are highly susceptible to the attack of various pests, and yield declines therewith
(I.Nyamwasa.,2018；Corrêa et al.,2016).Also, it is sent out after quantifying to some regional crop yield losses
Existing, wherein it is especially big to influence accounting for pest, especially soil insect (Amoako-Atta, 1983；Sekamwate,2007；
Toepfer et al.,2014).After novel pesticide regulations are promulgated, investigates and deals with the use of illicit drug according to law and give criminal sanctions, China
Agricultural product security situation is gradually become better.But cannot be neglected be some areas vegetable pesticide residue over-standard phenomenon still than more serious
(Deng Bo etc., 2017).Have multinomial studies have shown that as the leek of green onion garlic class excessive pesticide residues especially severe (Luo Li,
2006).Pesticide Residues in Vegetables will generate various harm more than a certain amount of to human body, for example will lead to acute forms poisoning
Deng.Find environmentally friendly measure, instead of the chemical pesticide of related prevention and control pest and disease damage, have become there is an urgent need to.
Entomopathogenic nematode (Entomopathogenic nematode, EPN) is the specialization parasitism day of many insects
Enemy, can especially prevent and treat dwelling property of soil and borer pest.Typically belong to Steinernema Carpocapsae section (Steinernematidae) and different small bar
Two class of nematode section (Heterorhabditidae) (Shapiro-Ilan and Gaugler, 2002).And EPN is to non-target
Bio-safety does not pollute the environment, and will not generate resistance, can be mass-produced, and has become a kind of the alternative of great potential
Pesticide environmentally friendly biological control method (Patil et al., 2017；Ahmed et al.,2017).Several main EPN
The nearly several hundred kinds of undergrounds such as grub, root-knot nematode and ground harmful organism can be effectively prevented.EPN has become a kind of important
Biological control of insect pests natural enemy, have quite be widely applied, in sustainable pest management have huge application potential
(Patil et al.2017).EPN is public by the famous agricultural of more families as mature, environmentally friendly biological control product
Department's favor, is only second to Su Yun bacillus.
The infective stage larva (infective juveniles, IJ) of EPN is that it uniquely has infection ability and can in life
Free living is in the worm state outside pin main body, general diapause not feeding, can long-term surviving in soil, until finding targeted insect
(Luis et al.,2016).Symbiotic bacteria (such as Xenorhabdus and Photorhabdus) is carried in the enteric cavity of EPN, this
It is exactly the lethal pest reason for it of EPN (Shapiro-Ilan and Gaugler, 2002).IJ enters in insect bodies, some
Signal may induce nematode to restore feeding for food signal and continue to develop, this process is known as the recovery of infection phase nematode,
Then EPN discharges symbiotic bacteria in host's haemocoele, these bacteriums breed rapidly, provides suitable ring for elegans development and breeding
Border, the host tissue of nematode then trophobiosis bacterium and degradation, and the lethal host within 48 hours, EPN are numerous in pin main body
Grow 1-3 generation, finally release a large amount of IJ, continually look for and infect other hosts (Poinar&Thomas, 1966；Shapiro-
Ilan et al.,2014)。
Using EPN carry out insect biological control, first to overcome the problems, such as be exactly mass production EPN (Glaser,
1931).At present EPN (Shapiro-Ilan can be produced using three kinds of living insects culture, solid culture, Liquid Culture methods
et al.,2016)。
Living insects culture nematode has the characteristics that energetic, sustained release, high-efficient really.But insect in incubation
Worm corpse is easy to be destroyed by eye fly larva, leads to problems such as nematode that can not grow.In addition, in insect living body incubation, existing
Insect itself is at high cost, insect cocoons needs repeatedly peeling etc. manually investment is high, insect infection rate is relatively low, insect bodies adhesion
The problems such as (Shapiro-Ilan et al., 2016), lead to the at high cost of living body culture nematode, it is higher to be only limitted to added value
The fields such as facilities vegetable, characteristic fruit tree, production is difficult to standardize and high cost seriously restricts the extensive use of the product.Using liquid
Body ferments large-scale culture tank fermentation technique (10~30 cubic metres of fermentors) (Shapiro-Ilan and Gaugler, 2002),
Cost is relatively low.But this method technical requirements are high, early investment is big, high energy consumption, production procedure mostly complicated, incubation
Once microbiological contamination is disposably lost larger in, before market is without sufficiently expanding, it is difficult to attract investment to found the factory and promote, temporarily not
It is suitable for China's national situation.In addition, in the liquid large-scale production nematode later period, need a large amount of water cleaning to collect nematode, generate a large amount of useless
Water, wastewater treatment bring environmental protection cost also rises suddenly while resulting in waste of resources.
In contrast, solid culture nematode technology, need place is small, investment is low, production link is few, energy consumption is low, production mark
Standardization is very suitable to China's national situation, is worth greatly developing and promote.Solid culture technology generally uses cheap animal (chicken, fish)
Internal organ and artificial formulation media (Ramakuwela et al., 2016), it is only necessary to autoclave, superclean bench and similar supermarket
The culturing rack of shelf, cumulative investment are no more than 200,000 yuans, are very suitable to found the factory on the spot and apply on the spot, decrease long-distance
The cost and risk of low-temperature transport.Moreover, new production method uses batch cultur bag, it is several during nematode solid culture
Waste water and waste material are not generated, environmentally friendly friendly production method is belonged to.
Solid culture is initially two dimensional model, and Bedding is by being added sponge as culture medium media implementation culture medium
Threedimensional model, use till today always.Originally solid medium is the substance based on livestock such as dog food, Pigs Kidney, ox blood, after
To develop to the inexpensively more stable substance such as yeast extract, nutrient broth, vegetable oil, soy meal, egg yolk, egg white
(Leite L G et al.,2016).The suitable condition of in vitro culture of selection and the suitable culture medium of screening are to nematode culture
Period, ultimate output and quality play a crucial role (R Gaugler.1999,2001；Yoo SK 2000).Many is ground
The person of studying carefully has done numerous studies to determine the critical nutrients factor of in vitro culture nematode, such as protein source and Lipid sources
(Shapiro-Ilan et al.,2014；Leite L G et al., 2016), specifically can use in animal (such as chicken, fish)
Dirty, animal matrix (such as peptone, yolk, milk), vegetable oil (such as peanut oil, corn oil, rapeseed oil).But in these people
The nematode produced on work culture medium may be compared with the nematode poor quality produced in insect bodies.If researcher has found artificial medium
If there is no similitude with insect host, the nematode poor quality (Womersley, 1993) produced.Abu Hatab M and R
The lipid that Gaugler compares the nematode produced in vivo and in vitro is constituted, it is found that culture medium and cultural method significantly affect nematode
Lipid mass, the lipid of insect is added in artificial medium and cultivates nematode, offspring's nematode lipid constitute and In vivo culture
Nematode lipid constitute it is much like, and improve nematode developmental rate and yield (Abu Hatab M and R Gaugler.,
2001).The artificial medium of discovery addition insect host can obtain preferable nematode growth rate, higher nematode production again later
With quality (Hatab and Gaugler 1999).
Nematode is commercially produced nearly 30 years, but the successful application of nematode is limited (Leite et al., 2016), phase
Compared with chemical insecticide, the high cost and production process unstability of nematode hinder the extensive use (Shapiro- of nematode
Ilan and Gaugler 2002).In recent years, optimization nematode production (Leite et al.2017) is continuously attempted in the world.
Existing solid culture based formulas does not add host insect powder generally, for example, Leite optimization one liter of system training
Support base basic components are as follows: glucose 25g, yeast extract 23g, yolk 6.25g, egg white 6.25g, sodium chloride 5g, agar 2g, flower
Oil generation 40g.The nematode produced on the artificial medium that these do not add host insect powder may be compared with the line produced in insect bodies
Worm poor quality.If researcher has found that artificial medium does not have similitude with insect host, the nematode poor quality produced
(Womersley,1993)。
EPN and insect pass through long-term coevolution, and nematode has been able to maximally utilize insect and nourishes and generates offspring.Have
Studies have shown that insect lymph can promote to infect the recovery rate (Bai and Grewal, 2007) of phase nematode.But addition elder brother
The research of worm powder is rarely reported.It is presumed that in insect bodies, there may be some active materials or suitable nutriment, energy
The yield and vigor of nematode are enough significantly improved, production cost reduces, and increases the synthesized competitiveness of nematode.
Summary of the invention
Problems to be solved by the invention
An object of the present invention is to provide a kind of raising solids manufacture entomopathogenic nematode yield
Culture medium.Using culture medium of the present invention can be improved solids manufacture entomopathogenic nematode efficiency,
It saves the production time, reduce production cost.The nematode produced by culture medium of the present invention can significantly add
The death of fast prodenia litura, improves the quality of nematode, enhances the insecticidal power of nematode.
It is another object of the present invention to provide a kind of culture mediums for preparing raising solids manufacture entomopathogenic nematode yield
Preparation method and applications.
The solution to the problem
The present inventor conducts in-depth research, repetition test in view of above-mentioned problems of the prior art, and discovery is logical
The Yellow meal worm larva powder that the freeze-drying of 5% concentration is added in the culture of basis is crossed, so as to complete the present invention.That is, of the invention
It is as described below:
Present invention firstly provides a kind of for improving the nematode culture medium of solids manufacture entomopathogenic nematode yield, and feature exists
In, in the nematode culture medium include following components: (1) basal medium, and (2) 5% concentration freeze-drying yellow meal worm
Larva powder, wherein serum-free in the basal medium, and do not include pluck and milk powder in the nematode culture culture medium
Equal lipid materials.The wherein composition of the basal medium are as follows: glucose 25g/L, yeast extract 23g/L, egg yolk
6.25g (other birds yolk can also substitute), egg white 6.25g (other birds egg white can also substitute), sodium chloride 5g/L,
Agar 2g/L, peanut oil 40g/L are settled to proper volume with distilled water.Preferably, the yolk is egg yolk, the egg white
For egg white.
Nematode solid medium of the present invention, wherein the preferred concentration of Yellow meal worm larva powder of the freeze-drying is
3wt%~5wt%, preferred concentration are 5wt%.The preparation method of the Yellow meal worm larva powder of heretofore described freeze-drying
Are as follows: fresh and alive Yellow meal worm larva is subjected to freezing processing, then carries out freezing dry-cure using vacuum freeze drier, then grind
Cheng Fen.
The present invention also provides a kind of methods for preparing nematode solid culture culture medium, it is characterised in that: in basis training described above
The Yellow meal worm larva powder for adding the freeze-drying of 1wt%~5wt% in base is supported, appropriate sponge is added, stirs evenly, in 121 after sealing
DEG C high pressure steam sterilization 15 minutes.The wherein additive amount of the sponge are as follows: 2~3g sponge is added in every 50ml basal medium.
The present invention further provides a kind of application of nematode solid medium in culture nematode.It is characterized in that, will be through excessively high
After culture medium cooled and solidified described above after pressing steam sterilizing, it is inoculated with entomopathogenic nematode symbiotic bacteria, at 22~28 DEG C
It is cultivated in incubator；After 1~3 day, it is inoculated with sterile nematode, is cultivated in 22~28 DEG C of incubators, collects nematode after 10~14 days.
Wherein the cultivation temperature of the incubator is 25 DEG C, and the sterile nematode is the sterile nematode of the egg hatching of culture two days.Wherein
The inoculum density of the nematode are as follows: 100 nematodes/milliliter solid medium.
The effect of invention
By technical solution of the present invention as it can be seen that culture medium of the invention compared with prior art, has the advantages that
1) it is raw to be added to solid using the Yellow meal worm larva powder of the freeze-drying of 5% concentration as one-component for the first time by the present invention
In the basal medium for producing entomopathogenic nematode, effectively increases the efficiency of solids manufacture entomopathogenic nematode, saved production
Time, reduction produce cost.Experiments have shown that the nematode produced by culture medium of the present invention can dramatically speed up prodenia litura
Death, improve the quality of nematode, enhance the insecticidal power of nematode.
2) useful supplement of culture medium of the invention as the culture medium of solids manufacture entomopathogenic nematode, improves nematode
The scope of application of culture medium.
3) yellow meal worm powder of 5% (2.5g) is added in 50mL basal medium, yield about improves 1 times, nematode desinsection
Effect enhancing, but the cost of the yellow meal worm powder of 2.5g only has 0.35 yuan, greatly reduces production cost, and elder brother is widely applied to realize
Worm pathogenic nematode biological insecticides are had laid a good foundation.
In order to which above and other objects, features and advantages of the invention can be clearer and more comprehensible, below special lift preferably implement
Example, and cooperate Figure of description, it is described in detail below:
Detailed description of the invention
Fig. 1 is the functional study flow chart of the culture of entomopathogenic nematode in-vitro solid and the culture medium of powder containing Yellow meal worm larva；
Fig. 2-1 and Fig. 2-2 is respectively test for the first time and second of test solid culture nematode production situation.Wherein, CK
For control group, represents and do not add yellow meal worm powder in solid medium；T1%, T3% and T5% respectively represent solid medium addition
1%, the yellow meal worm powder of 3%, 5% concentration；Different letters indicate the different conspicuousnesses (P≤0.05) between different disposal；
Fig. 3-1 and Fig. 3-2 is respectively that test for the first time and second of test solid culture nematode infection prodenia litura are lethal
Time situation.Wherein, CK is control group, and yellow meal worm powder is not added in representative in solid medium；T1%, T3% and T5% difference
Represent the yellow meal worm powder that solid medium adds 1%, 3%, 5% concentration；Different letters indicate that the difference between different disposal is aobvious
Work property (P≤0.05).
Specific embodiment
Embodiment 1: the preparation of the culture medium of powder containing Yellow meal worm larva
The preparation of 1.1 basal mediums
The formula of the basal medium of the 1L system of this solid culture are as follows: glucose 25g, yeast extract 23g, egg yolk
6.25g (other birds yolk can also substitute), egg white 6.25g (other birds egg white can also substitute), sodium chloride 5g, fine jade
Rouge 2g, peanut oil 40g, addition 1L distilled water are settled to 1L.
1L basal medium is prepared according to above-mentioned formula, average mark is filled in the conical flask of 250mL, each 250mL taper
Bottle packing basal medium 50mL.
The preparation of the nematode culture medium of the 1.2 freeze-drying Yellow meal worm larva powder containing various concentration
It buys Yellow meal worm larva (market purchasing), freezing is freeze-dried on vacuum freeze drier for 24 hours, after dry
Yellow meal worm larva powder is worn into, is saved in -20 DEG C of refrigerators stand-by.
The freeze-drying Yellow meal worm larva powder that 2.5g is added in the basal medium that above-mentioned 1.1 section is prepared, to obtain
Nematode culture medium comprising the freeze-drying Yellow meal worm larva powder that mass volume ratio is 5%, and 2.4g is added (volume is that 1cm3 is left
It is right) sponge (bought by ten thousand happy purchase shops of online shopping mall of Taobao, high density yellow sponge, 35 yuan/6cm X 30cm X
30cm), 121 DEG C high pressure steam sterilization 15 minutes after sealing are stirred evenly.Solids manufacture entomopathogenic nematode is improved to be prepared
The culture medium of yield.
Referring to the preparation method of above-mentioned culture medium, prepared respectively without freeze-drying Yellow meal worm larva powder (as control
Group), mass volume ratio be the 1% freeze-drying Yellow meal worm larva powder of 0.5g (i.e. in 50ml basal medium add) and quality
Volume ratio is the Yellow meal worm larva powder of 3% (the freeze-drying Yellow meal worm larva powder of 1.5g is added i.e. in 50ml basal medium)
Nematode culture medium.
Embodiment 2: the functional study of the Yellow meal worm larva powder culture medium containing freeze-drying
The acquisition of 2.1 entomopathogenic nematode (abbreviation EPN) symbiotic bacterias
(1) entomopathogenic nematode (abbreviation EPN) symbiotic bacteria isolates and purifies
EPN infects about 0.5g greater wax moth and (purchased from Tianjin Hui Yude Biotechnology Co., Ltd, and is stored in 14 DEG C of laboratory
In biochemical cultivation case): prepare 24 orifice plates, the great-hearted greater wax moth larva of a health is placed in every hole.Sf nematode solution is taken to estimate
Its density guarantees that 100 nematodes are added in each hole, is placed in 25 DEG C of cultures.30h-36h or so, under superclean bench, from 24
Orifice plate takes out greater wax moth corpse and is put into 75% alcohol and carries out surface disinfection, and second pair of abdominal foot of greater wax moth is cut, with primary
Property oese dip insect lymph plate streak be seeded to NBTA differential medium (formula: 33 grams of nutrient agar, in bromine hundred
Blue 0.025 gram, 0.04 gram of red tetrazolium, 1000 milliliters of distilled water of phenol) on cultivated in 25 DEG C.
It will appear red and two kinds of bacterium colonies of blue-green on culture medium two days later, picking blue-green colony inoculation to new NBTA is trained
It supports on base, until all blue-green (i.e. I type bacterium) is presented in the bacterium colony on culture medium.Picking single bacterium falls within TSB+Y culture solution
In (formula: 40 grams of pancreas peptone soybean broth, 5 grams of yeast extract, distilled water 1000mL), 25 DEG C, 200rpm shaking table culture
24h。
Bacterium solution after coated plate is verified is placed after a week in 4 DEG C of refrigerators, and the sterile glycerol for being 30% with concentration is according to 1:1's
Volume ratio mixing is placed in bacterium freezen protective pipe, and is stored at -80 DEG C.
(2) EPN infects greater wax moth, and White trap method collects EPN worm sources
Prepare 24 orifice plates, the filter paper dick close with aperture diameter is placed in hole, a health is placed in each hole work
The greater wax moth larva of power.S.feltiae nematode (genus steinernema) solution is taken, density is estimated, guarantees that 100 are added in each hole
Nematode, 25 DEG C of cultures.Greater wax moth is dead after 48h.Greater wax moth corpse is transferred to White trap device, concrete operations are as follows: will
The 6cm culture dish lid of covering wet filter paper is just being put into 9cm culture dish, outside 6cm culture dish lid, in 9cm culture dish
3mL sterile water is added, greater wax moth corpse is placed in 6cm culture dish lid, finally seals up 9cm culture dish with sealed membrane.One
EPN is collected after or so week, is stored in 14 DEG C, and use in two weeks.
2.2 nematode inoculation methods
After prepared nematode culture medium is cooling in example 1 to be performed, under superclean bench into every bottle of nematode culture medium
It is inoculated with the 1mL2.1 section entomopathogenic nematode symbiotic bacteria, is placed in stationary culture under the conditions of 25 DEG C of temperature.After 2 days,
It is inoculated with the sterile nematode of the egg hatching of 5000 cultures two days into each conical flask, is finally cultivated 2 weeks in 25 DEG C of incubators
Collect (aforesaid operations process is referring to Fig. 1) afterwards.
Influence of the 2.3 Yellow meal worm larva powder for nematode
Test be divided into control group (do not add freeze-drying Yellow meal worm larva powder) and experimental group (add 1wt%, 3wt%,
The freeze-drying Yellow meal worm larva powder of 5wt%), 2 tests are carried out altogether, and test is repeated 4 times every time, is averaged and is tied as test
Fruit.
(1) nematode is collected, yield is measured
Collect nematode: EPN is cultivated after two weeks, and the sponge in conical flask is poured into nematode collection device, collection device is set
In large beaker, 1075mL tap water is added, presses sponge 60 times, water is poured into bucket.It is repeated 4 times altogether.It is molten by what is be collected into
Liquid uniform stirring takes out 25mL and is stored in Tissue Culture Flask.
Measurement yield trials: the EPN solution in Tissue Culture Flask is placed in 50mL beaker equal with magnetic stirrer
It is even, it takes 5mL solution to be placed in 45mL tap water and is counted after 10 times of dilution, amount to number twice.Calculate yield.
(2) virulence is tested
Prodenia litura is cultivated, the great-hearted Spodoptera litura larvae of health for selecting similar weight (about 0.5g) is tested.
Lethality test: preparing 24 orifice plates, the filter paper dick close with aperture diameter be placed in hole, and each hole places one
Spodoptera litura larvae.S.feltiae nematode (genus steinernema) nematode solution 100 (volume is 50 μ L) is taken to be added in orifice plate
Each hole, 25 DEG C culture.Death condition is recorded every 8h.
(3) experimental result and discussion (see Fig. 1 and Fig. 2)
[influence of the Yellow meal worm larva powder to nematode production]:
It repeats to test for the first time, control group nematode average product is 2.95 X 106Item/bottle, addition 1%, 3% and 5% are dense
The nematode average product of the yellow meal worm powder processing of degree is 4.25 X 10 respectively6Item/bottle, 4.97 X 106Item/bottle, 7.49 X 106Item/
Bottle.It can be seen that addition 1wt%, the nematode production of the yellow meal worm powder processing of 3wt% and 5wt% concentration is all significantly higher than control
The nematode production (df=3, F=16.31, P≤0.001) of group, with the increase of concentration, nematode production is accordingly increased, especially
The nematode production highest for adding the yellow meal worm powder processing of 5wt% concentration, be control group yield 2.54 times (referring specifically to Fig. 2-1,
Wherein, abscissa C represents control group；T1%, T%3 and T5% respectively represent the Yellow meal worm larva of 1wt%, 3wt% and 5wt%
Powder).
Second of repetition is tested, and control group nematode production is 2.01 X 106Item/bottle, 1%, 3% and 5% concentration of addition
The nematode production of yellow meal worm powder processing is 2.80 X 10 respectively6Item/bottle, 4.24 X 106Item/bottle, 4.81 X 106Item/bottle.Thus
As it can be seen that the nematode production of the yellow meal worm powder processing of addition 3wt% and 5wt% concentration is all significantly higher than the nematode production of control group
(df=3, F=7.151, P=0.008), with the increase of concentration, nematode production is accordingly increased, especially addition 5wt% concentration
Yellow meal worm powder processing nematode production highest, be 2.40 times (referring specifically to Fig. 2-2) of control group yield.
It is sufficiently proved by above-mentioned double repeated experiment: the solid culture of the yellow meal worm powder of addition 1wt%~3wt% concentration
Base can be improved the yield of nematode, wherein the solid medium of addition 3wt% and 5wt% yellow meal worm powder can significantly improve nematode
Yield, and can be improved the efficiency of solids manufacture entomopathogenic nematode, and then saved the time of culture nematode, reduce production
Cost.
[influence of the yellow meal worm powder to nematode lethality]:
It repeating to test for the first time, prodenia litura average death time is 33.2h after control group nematode infection, 1wt% is added,
3wt% and 5wt% concentration yellow meal worm powder processing nematode infection after prodenia litura average death time be respectively 31.2h,
31.2h and 28h.It can be seen that the prodenia litura average death time of the yellow meal worm powder processing of addition 5wt% concentration is considerably shorter than
The prodenia litura average death time (df=3, F=8.439, P=0.004) (referring specifically to Fig. 3-1) of control group.
Second of repetition is tested, and prodenia litura average death time is 37h after control group nematode infection, adds 1wt%,
Prodenia litura average death time after the nematode infection of the yellow meal worm powder processing of 3wt% and 5wt% concentration is respectively 32h, 30h
And 26.8h, with the increase of concentration, prodenia litura average death time is reduced, at the yellow meal worm powder for adding 3% and 5% concentration
The prodenia litura average death time of reason is all considerably shorter than prodenia litura average death time (df=3, the F=of control group
8.439, P=0.004) (referring specifically to Fig. 3-2).
Sufficiently prove by above-mentioned double repeated experiment: the nematode of the solid medium production of addition 5wt% yellow meal worm powder can show
The death for accelerating prodenia litura is write, the quality of nematode is improved, enhances the insecticidal power of nematode.
Specific embodiments of the present invention are described in detail above, but it is only used as example, the present invention is not intended to limit
In particular embodiments described above.To those skilled in the art, it is any to the invention carry out equivalent modifications and replace
In generation, is also all among scope of the invention.Therefore, without departing from the spirit and scope of the invention made by equal transformation and repair
Change, all should be contained within the scope of the invention.
Claims (10)
1. a kind of nematode solid medium, which is characterized in that the culture medium includes following components: (1) basal medium and (2)
The Yellow meal worm larva powder of the freeze-drying of 1wt%~5wt%, wherein the basal medium serum-free, and the nematode solid
The lipid materials such as pluck and milk powder are not included in culture medium.
2. nematode solid medium according to claim 1, wherein the basal medium forms are as follows: glucose 25g/L,
Yeast extract 23g/L, yolk 6.25g, egg white 6.25g, sodium chloride 5g/L, agar 2g/L, peanut oil 40g/L, and with distillation
Water is settled to proper volume；Preferably, the yolk is egg yolk, and the egg white is egg white.
3. nematode solid medium according to claim 1 or 2, wherein the Yellow meal worm larva powder of the freeze-drying is preferred
Concentration be 3wt%~5wt%, preferred concentration be 5wt%.
4. nematode solid medium described in any one of claim 1 to 3, wherein the yellow meal worm children of the freeze-drying
Worm powder the preparation method comprises the following steps: fresh and alive Yellow meal worm larva is carried out freezing processing, it is then cold using vacuum freeze drier progress
Frozen dried, then pulverize.
5. a kind of method for preparing any one of Claims 1 to 4 nematode solid culture culture medium, it is characterised in that: in institute
The Yellow meal worm larva powder for adding the freeze-drying of 1wt%~5wt% in basal medium is stated, appropriate sponge is added, stirs evenly, is sealed
Afterwards in 121 DEG C high pressure steam sterilization 15 minutes.
6. according to the method described in claim 5, the wherein additive amount of the sponge are as follows: add 2 in every 50ml basal medium
~3g sponge.
7. application of the described in any item nematode solid mediums of Claims 1 to 4 in culture nematode.
8. application according to claim 7, which is characterized in that by the culture medium cooled and solidified after high pressure steam sterilization
Afterwards, it is inoculated with entomopathogenic nematode symbiotic bacteria, is cultivated in 22~28 DEG C of incubators；After 1~3 day, it is inoculated with sterile nematode, 22
It is cultivated in~28 DEG C of incubators, collects nematode after 10~14 days.
9. application according to claim 8, which is characterized in that wherein the cultivation temperature of the incubator is 25 DEG C, described
Sterile nematode is the sterile nematode of the egg hatching of culture two days.
10. application according to claim 8 or claim 9, wherein the inoculum density of the nematode are as follows: 100 nematodes/milliliter solid
Culture medium.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110973066A (en) * | 2019-12-05 | 2020-04-10 | 南京林业大学 | Culture medium for wood-rotting nematodes infected with pine wood nematode disease and preparation method and application thereof |
| CN110973072A (en) * | 2019-12-30 | 2020-04-10 | 吉林农业科技学院 | Method for collecting large amount of pure eggs of clonorchis sinensis |
Citations (9)
| 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 |
| CN1644035A (en) * | 2004-12-02 | 2005-07-27 | 赵昕 | Culture of nematoda without bacteria |
| CN101157893A (en) * | 2007-09-11 | 2008-04-09 | 中国科学院东北地理与农业生态研究所 | Large scale insect pathogenic nematodes living high-power culturing method |
| CN102796187A (en) * | 2011-05-27 | 2012-11-28 | 中国科学院上海生命科学研究院 | New method for pest control on basis of ribonucleic acid interfere (RNAi) technology |
| CN102972446A (en) * | 2012-11-22 | 2013-03-20 | 北京市西山试验林场 | Preparation method of steinernema carpocapsae |
| KR20140092525A (en) * | 2013-01-15 | 2014-07-24 | 주식회사 에스엠바이오비전 | The culture medium composition for insect infectious nematodes which comprising Tenebrio molitor Linnaeus Meal Worm and vermiculite |
| CN104039755A (en) * | 2011-09-01 | 2014-09-10 | 百奥泰利加控股有限公司 | Insecticidal lipid agents isolated from entomopathogenic fungi and uses thereof |
| CN106305626A (en) * | 2016-08-18 | 2017-01-11 | 浙江绿神天敌生物技术有限公司 | Artificial culture medium for entomopathogenic nematodes |
| CN106358858A (en) * | 2016-08-28 | 2017-02-01 | 新疆农业科学院微生物应用研究所 | Application of heterorhabditis bacteriophora to biological control of underground insects of greenhouse vegetables |
-
2018
- 2018-10-15 CN CN201811194454.1A patent/CN109169534A/en active Pending
Patent Citations (9)
| 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 |
| CN1644035A (en) * | 2004-12-02 | 2005-07-27 | 赵昕 | Culture of nematoda without bacteria |
| CN101157893A (en) * | 2007-09-11 | 2008-04-09 | 中国科学院东北地理与农业生态研究所 | Large scale insect pathogenic nematodes living high-power culturing method |
| CN102796187A (en) * | 2011-05-27 | 2012-11-28 | 中国科学院上海生命科学研究院 | New method for pest control on basis of ribonucleic acid interfere (RNAi) technology |
| CN104039755A (en) * | 2011-09-01 | 2014-09-10 | 百奥泰利加控股有限公司 | Insecticidal lipid agents isolated from entomopathogenic fungi and uses thereof |
| CN102972446A (en) * | 2012-11-22 | 2013-03-20 | 北京市西山试验林场 | Preparation method of steinernema carpocapsae |
| KR20140092525A (en) * | 2013-01-15 | 2014-07-24 | 주식회사 에스엠바이오비전 | The culture medium composition for insect infectious nematodes which comprising Tenebrio molitor Linnaeus Meal Worm and vermiculite |
| CN106305626A (en) * | 2016-08-18 | 2017-01-11 | 浙江绿神天敌生物技术有限公司 | Artificial culture medium for entomopathogenic nematodes |
| CN106358858A (en) * | 2016-08-28 | 2017-02-01 | 新疆农业科学院微生物应用研究所 | Application of heterorhabditis bacteriophora to biological control of underground insects of greenhouse vegetables |
Non-Patent Citations (4)
| Title |
|---|
| MOEEN A. ABU HATAB1 AND RANDY GAUGLER: "Lipids of in Vivo and in Vitro Cultured Heterorhabditis bacteriophora", 《BIOLOGICAL CONTROL》 * |
| SHIYU ZHEN 等: "Enhanced entomopathogenic nematode yield and fitness via addition of pulverized insect powder to solid media", 《JOURNAL OF NEMATOLOGY》 * |
| 白向宾: "《中国优秀硕士学位论文全文数据库农业科技辑》", 15 February 2016 * |
| 郝杰 等: "一种新的昆虫病原线虫固体培养基", 《中国植保导刊》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110973066A (en) * | 2019-12-05 | 2020-04-10 | 南京林业大学 | Culture medium for wood-rotting nematodes infected with pine wood nematode disease and preparation method and application thereof |
| CN110973066B (en) * | 2019-12-05 | 2021-08-17 | 南京林业大学 | Culture medium for wood-rotting nematodes infected with pine wood nematode disease and preparation method and application thereof |
| CN110973072A (en) * | 2019-12-30 | 2020-04-10 | 吉林农业科技学院 | Method for collecting large amount of pure eggs of clonorchis sinensis |
| CN110973072B (en) * | 2019-12-30 | 2021-08-31 | 吉林农业科技学院 | Method for collecting large amount of pure eggs of clonorchis sinensis |
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