CN105766813A - Method for gathering and cultivating aphelenchoidessp - Google Patents
Method for gathering and cultivating aphelenchoidessp Download PDFInfo
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- CN105766813A CN105766813A CN201610228429.5A CN201610228429A CN105766813A CN 105766813 A CN105766813 A CN 105766813A CN 201610228429 A CN201610228429 A CN 201610228429A CN 105766813 A CN105766813 A CN 105766813A
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- 238000000034 method Methods 0.000 title claims abstract description 39
- 239000002689 soil Substances 0.000 claims abstract description 47
- 241000233866 Fungi Species 0.000 claims abstract description 11
- 241000244206 Nematoda Species 0.000 claims description 72
- 239000005645 nematicide Substances 0.000 claims description 45
- 239000001963 growth medium Substances 0.000 claims description 33
- 230000001954 sterilising effect Effects 0.000 claims description 26
- 241000123650 Botrytis cinerea Species 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 12
- 238000004659 sterilization and disinfection Methods 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 210000001015 abdomen Anatomy 0.000 claims description 9
- 238000012258 culturing Methods 0.000 claims description 9
- YPHMISFOHDHNIV-FSZOTQKASA-N cycloheximide Chemical compound C1[C@@H](C)C[C@H](C)C(=O)[C@@H]1[C@H](O)CC1CC(=O)NC(=O)C1 YPHMISFOHDHNIV-FSZOTQKASA-N 0.000 claims description 8
- 239000012528 membrane Substances 0.000 claims description 8
- 230000008569 process Effects 0.000 claims description 8
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 6
- 230000000056 copulatory effect Effects 0.000 claims description 6
- 210000003238 esophagus Anatomy 0.000 claims description 6
- 238000012797 qualification Methods 0.000 claims description 6
- 238000000926 separation method Methods 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 5
- WEEMDRWIKYCTQM-UHFFFAOYSA-N 2,6-dimethoxybenzenecarbothioamide Chemical compound COC1=CC=CC(OC)=C1C(N)=S WEEMDRWIKYCTQM-UHFFFAOYSA-N 0.000 claims description 4
- 229960002385 streptomycin sulfate Drugs 0.000 claims description 4
- 241001260012 Bursa Species 0.000 claims description 3
- 241000628997 Flos Species 0.000 claims description 3
- 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 3
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 3
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims description 3
- 229910000831 Steel Inorganic materials 0.000 claims description 3
- WYTGDNHDOZPMIW-RCBQFDQVSA-N alstonine Natural products C1=CC2=C3C=CC=CC3=NC2=C2N1C[C@H]1[C@H](C)OC=C(C(=O)OC)[C@H]1C2 WYTGDNHDOZPMIW-RCBQFDQVSA-N 0.000 claims description 3
- 210000003323 beak Anatomy 0.000 claims description 3
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 3
- 210000004907 gland Anatomy 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 230000000968 intestinal effect Effects 0.000 claims description 3
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 claims description 3
- 229920005610 lignin Polymers 0.000 claims description 3
- 235000012054 meals Nutrition 0.000 claims description 3
- 235000013379 molasses Nutrition 0.000 claims description 3
- 230000003287 optical effect Effects 0.000 claims description 3
- 208000005368 osteomalacia Diseases 0.000 claims description 3
- 239000001103 potassium chloride Substances 0.000 claims description 3
- 235000011164 potassium chloride Nutrition 0.000 claims description 3
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- 230000007480 spreading Effects 0.000 claims description 3
- 238000003892 spreading Methods 0.000 claims description 3
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- 235000013305 food Nutrition 0.000 abstract description 11
- 230000008901 benefit Effects 0.000 abstract description 3
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- 238000011081 inoculation Methods 0.000 description 3
- 230000001228 trophic effect Effects 0.000 description 3
- 241000274183 Micromeria Species 0.000 description 2
- 244000061176 Nicotiana tabacum Species 0.000 description 2
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 2
- 238000012271 agricultural production Methods 0.000 description 2
- 229910052785 arsenic Inorganic materials 0.000 description 2
- RQNWIZPPADIBDY-UHFFFAOYSA-N arsenic atom Chemical compound [As] RQNWIZPPADIBDY-UHFFFAOYSA-N 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
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- 241000894007 species Species 0.000 description 2
- 239000002028 Biomass Substances 0.000 description 1
- 241000269799 Perca fluviatilis Species 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
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- 230000005110 hydrotaxis Effects 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
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- 208000015181 infectious disease Diseases 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
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- 230000000877 morphologic effect Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 239000001965 potato dextrose agar Substances 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
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- 238000003900 soil pollution Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
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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)
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- Biodiversity & Conservation Biology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention belongs to the field of biotechnologies, and particularly discloses a method for gathering and cultivating aphelenchoidessp. The method particularly includes (1), separating the aphelenchoidessp; (2), identifying the aphelenchoidessp; (3) disinfecting the surfaces of the aphelenchoidessp; (4), gathering and cultivating the aphelenchoidessp. The method has the advantages that the method is simple and feasible, and the aphelenchoidessp is high in propagation survival rate, good in individual quality, little in heredity change and convenient to manage; a large quantity of the purely cultivated aphelenchoidessp can be effectively obtained by the aid of the method, and technical support can be provided for researching key functions of the aphelenchoidessp in integral food chains of soil and interaction of the aphelenchoidessp and fungi on the growth of plants.
Description
Technical field
The invention belongs to biological technical field, be specifically related to the method that the sliding sword of a kind of enrichment culture belongs to Fungal-feeding nematode.
Background technology
In soil, the nematicide of perch has comparative advantage with free-living nematode, and soil free-living nematode is based on bacterial-feeding nematodes and Fungal-feeding nematode (both are referred to as food Micronema), accounts for the 80% of free-living nematode.A lot of researchs show, food Micronema, by preying on soil microorganism, accelerates microbial reproduction, and by the nutrient that metabolic secretion and releasing microbe hold, and then affect soil ecological process and plant growing.But this effect can be different because of the C/N nutriture of the kind of different nematicides, microorganism and plant and soil matrix.Interacting and the report of plant growing is less currently, with respect to soil Fungal-feeding nematode and fungus, the especially rare report especially of the correlational study in contaminated soil.Such research can further investigate feeding habit and the trophic behaviour critical role in soil food chain and whole soil nutrient circulate thereof of soil Fungal-feeding nematode, and the soil Fungal-feeding nematode of pure culture is the basis of research.Many research show sliding sword belong to Fungal-feeding nematode (AphelenchoidesSp.) soil pollution is had stronger patience, be widely present in all kinds of contaminated soil, its further investigation is significant.But, rarely having the Enrichment culture method belonging to Fungal-feeding nematode about the especially sliding sword of soil Fungal-feeding nematode at present, technical difficult points is in that the qualification (especially identification of living body) of soil Fungal-feeding nematode needs personnel to have relevant speciality background and working experience for many years；The enrichment culture of soil Fungal-feeding nematode needs fungal species, kinds of culture medium and formula, inoculation opportunity, condition of culture etc. are carried out long-term to grope research；The preservation condition of soil Fungal-feeding nematode and renewal time are also to ensure that nematicide can essential condition long-term, efficient, prolific.The invention provides the sliding sword of a kind of soil and belong to the separation of Fungal-feeding nematode, qualification and Enrichment culture method, the phytoremediation for soil biology research, agricultural production and contaminated soil provides technical support.The patent CN103688908A declared before inventor provides the sliding sword of a kind of enrichment culture and belongs to the Enrichment culture method of Fungal-feeding nematode, the method achieves certain effect, but the nematicide rate of increase need nonetheless remain for further raising, to adapt to the application demand of agricultural pollution soil remediation.
Summary of the invention
It is an object of the invention to provide the sliding sword of a kind of enrichment culture belong to Fungal-feeding nematode (AphelenchoidesSp.) method, the method is easy to use, and the breeding potential that sliding sword belongs to Fungal-feeding nematode is high, can belong to Fungal-feeding nematode offer technical guarantee by the sliding sword for efficiently preparing a large amount of pure culture.
The method that the sliding sword of a kind of enrichment culture provided by the invention belongs to Fungal-feeding nematode, specifically comprises the following steps that
1, slide sword belong to Fungal-feeding nematode (AphelenchoidesSp.) separation: from field acquisition fresh soil.The tray method improved is adopted to separate nematicide from soil.
2, slide sword belong to Fungal-feeding nematode (AphelenchoidesSp.) qualification: liquid in beaker is proceeded to culture dish, then belongs to the feature of Fungal-feeding nematode according to sliding sword and chooses this genus nematicide with capillary tube under anatomical lens, then it carries out identification of living body under an optical microscope.
3, slide sword belong to Fungal-feeding nematode (AphelenchoidesSp.) surface sterilization: proceeded to by nematicide in the centrifuge tube of sterilizing, carries out surface sterilization with streptomycin sulfate and cycloheximide mixed liquor to nematicide；
4, slide sword belong to Fungal-feeding nematode (AphelenchoidesSp.) enrichment culture: by fungus Botrytis cinerea (BotrytiscinereaPers.) it is inoculated in improved culture medium, adopts sealed membrane (Parafilm) to be sealed by culture dish, to prevent the evaporation of culture medium moisture and to pollute.Then culture dish is inverted and is put in constant incubator, 20-25 DEG C of dark culturing, after one week, when Botrytis cinerea amount reproduction, it is about to sliding sword belongs in the culture dish that uniformity Botrytis cinerea is covered with in nematicide access.Adopting sealed membrane (Parafilm) to be sealed by culture dish, culture dish faces up and puts into sizeable sterilizing box, 20-25 DEG C of dark culturing, whether routine observation culture dish is subject to the pollution of other funguses or acarid, after several weeks, ash spore disappears substantially, nematicide amount reproduction.Repetition above step can obtain substantial amounts of sliding sword and belong to Fungal-feeding nematode.Wherein, the formula of improved culture medium is: in every 1000ml improved culture medium, Rhizoma Solani tuber osi 120-150g, glucose 10-12g, fishbone powder 10-12g, conch meal 6-8 part, bean cake 3-5 part, lignin 3-5 part, molasses 2-3 part, disodium hydrogen phosphate 0.01-0.02 part, potassium chloride 0.01-0.02, ferrous sulfate heptahydrate 0.01-0.02 part, all the other are water, and pH value is natural.
The tray method equipment needed thereby of the improvement described in above-mentioned steps (1) is simple, operate easy, specific as follows: the rustless steel screen tray of 10 orders is put in supporting tray, screen tray is placed two-layer medicated napkin, then, soil sample uniform spreading on paper, add water until submergence soil in tray.It is placed under 25 DEG C of temperature, dark conditions again and separates.After 48h, collect nematicide in beaker with 400 mesh sieves.Stand after overnight, slowly draw supernatant liquid with vacuum pump, avoid disturbance lower floor liquid as far as possible.Now, lower floor's liquid have accumulated substantial amounts of nematicide.、
The major diagnostic feature that in above-mentioned steps (2), sliding sword belongs to Fungal-feeding nematode is as follows: 1. heat kill is after death, female worm polypide is until abdomen is curved, the male worm usual afterbody whole polypide of ventrad hook is " J " shaped (if live body is fubaritic, can determine after being killed, reference is provided for identification of living body), living nematode is many in serpentine；2. head is typically somewhat hung contracting, headstock osteomalacia, and lancet is relatively thin, and base portion expands, and length is generally 10-12 μm, is usually no more than 20 μm；3. esophagus presoma is cylindrical, the prosperity of middle oesophageal bulb, oval or square circular, and middle esophagus ball clack is flourishing, and rear esophageal gland leaf is flourishing, cover the back side of intestinal；4. porus excretorius is arranged in level place after oesophageal bulb；5. female worm tail is conical, tail end form of diverse: blunt circle or carefully justify, finger-like or bifurcated or have an abdomen to dash forward, with or without wake flowfield, the form of diverse of wake flowfield；6. male worm tail conical, to outside of belly hook；Copulatory spicules are Flos Rosae Rugosae thorn shape, and Ji Ding and base beak are generally flourishing, without copulatory bursa.
In above-mentioned steps (3), in described mixed liquor, the quality-volumetric concentration of streptomycin sulfate is 0.80-1.20g.L-1；The mass body volume concentrations of cycloheximide is 0.015-0.02g.L-1；The time that nematicide carries out surface sterilization is 15-20min.
In the present invention, the sterilized culture medium in step (4) is by culture medium in high-pressure sterilizing pot, and at 121 DEG C of temperature, sterilizing 30min obtains.The store method that the sliding sword obtained after its enrichment culture belongs to Fungal-feeding nematode is as follows: adopts sealed membrane (Parafilm) to be sealed by culture dish, is then saved in by culture dish in 4 DEG C of refrigerators standby.Every 5-6 month, sliding sword being belonged to Fungal-feeding nematode and carries out successive transfer culture, the scarcity of the evaporation of overlong time then culture medium moisture and food resource, nematicide can be dead, and the time is too short, is the waste of financial resources and labour.Its specific operation process is, culture dish is taken out from 4 DEG C of refrigerators, choose the culture medium of a wired worm of little block length, put in the modified culture medium covering with uniformity Botrytis cinerea, sizeable sterilizing box is put into after being sealed by culture dish, 20-25 DEG C of dark culturing, and whether routine observation culture dish be subject to the pollution of other funguses or acarid, is preserved as in 4 DEG C of refrigerators by culture dish after three weeks.
The beneficial effects of the present invention is: its method is simple, potato dextrose agar before has been improved, nutrition is comprehensive and abundant more, sliding sword is made to belong to Fungal-feeding nematode reproductive survival rate higher, nematicide Individual Quality is good, and heritability change is little, convenient management, the sliding sword that can effectively obtain a large amount of pure culture belongs to Fungal-feeding nematode, provides technical support for the trophic behaviour and whole soil food chain studying Fungal-feeding nematode in soil.In addition, sliding sword belongs to Fungal-feeding nematode and takes advantage in contaminated soil, its trophic behaviour to fungal mycelia can be utilized, accelerate the stubborn mineralising release of nutrient in mycelia or spore in contaminated soil, thus the growth of plant in contaminated soil can be promoted in agricultural production, in the reparation of contaminated soil, then can improve phytoremediation efficiency.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is described in further detail.
Embodiment 1
1, slide sword and belong to the separation of Fungal-feeding nematode: gather fresh soil from Institute of Botany's (Nanjing Botanical Garden Mem. Sun Yat-Sen) nursery lot.The tray method improved is adopted to separate nematicide from soil.Concrete grammar describes as follows.
The rustless steel screen tray of 10 orders is put in supporting tray, and on screen tray, places two-layer medicated napkin, then soil sample uniform spreading is sprinkling upon on paper, adds water until submergence soil in tray.Whole device is put in incubator, separates under 20～25 DEG C of temperature, dark conditions.Due to the hydrotaxis of nematicide, adding the effect of himself gravity, nematicide departs from soil successively and enters in water (be applicable to enlivening nematicide separation soil sample) through medicated napkin hole.After 48-60h, screen tray is taken out, the liquid in tray is poured into 400 mesh sieve dishes, collect nematicide after constantly rinsing screen tray with water in beaker.Beaker stands after overnight, slowly draws supernatant liquid with vacuum pump, and avoids disturbance lower floor liquid as far as possible.Now, lower floor's liquid have accumulated substantial amounts of nematicide.
2, slide sword and belong to the qualification of Fungal-feeding nematode
Rarely having Fungal-feeding nematode at present, especially sliding sword belongs to the report of Fungal-feeding nematode pure culture, and one of difficult point lies also in the qualification of nematicide, particularly enrichment culture process, it is necessary to by nematicide identification of living body；Authentication method is specific as follows: liquid in beaker proceeds to the culture dish of diameter 5cm.After standing 5min, belong to the morphological feature of nematicide according to sliding sword and choose this genus nematicide with capillary tube under anatomical lens, then by its identification of living body under Olympus Optical microscope.
Identifying that the major diagnostic feature that sliding sword belongs to foundation during Fungal-feeding nematode is as follows: 1. heat kill is after death, female worm polypide is until abdomen is curved, and the male worm usual afterbody whole polypide of ventrad hook is " J " shaped, and living nematode is many in serpentine；(if live body is fubaritic, can determine after being killed, and provides reference for identification of living body), 2. head is typically somewhat hung contracting, headstock osteomalacia, and relatively carefully, base portion expands lancet, and length is generally 10-12 μm, is usually no more than 20 μm；3. esophagus presoma is cylindrical, the prosperity of middle oesophageal bulb, oval or square circular, and middle esophagus ball clack is flourishing, and rear esophageal gland leaf is flourishing, cover the back side of intestinal；4. porus excretorius is arranged in level place after oesophageal bulb；5. female worm tail is conical, tail end form of diverse: blunt circle or carefully justify, finger-like or bifurcated or have an abdomen to dash forward, with or without wake flowfield, the form of diverse of wake flowfield；6. male worm tail conical, to outside of belly hook；Copulatory spicules are Flos Rosae Rugosae thorn shape, and Ji Ding and base beak are generally flourishing, without copulatory bursa.
3, slide sword and belong to the surface sterilization of Fungal-feeding nematode: nematicide is proceeded in the 50ml centrifuge tube of sterilizing, be 1.0gL by quality-volumetric concentration-1Streptomycin sulfate and quality-volumetric concentration be 0.02gL-1Cycloheximide mixed liquor nematicide is carried out surface sterilization 20min, 5000rmin-1Centrifugal 5min, removes supernatant, more repeatedly cleans with sterilized water 6 times.Fully shake when cleaning, 5000rmin every time-1Centrifugal 5min, abandoning supernatant.
4, slide sword and belong to the enrichment culture of Fungal-feeding nematode: Fungal-feeding nematode is main in soil makes a living taking food fungus, thus need to provide suitable food source to it in pure culture process.Fungal species is incorrect, then sliding sword belongs to Fungal-feeding nematode and is reluctant to take food, nematicide mortality；Type of culture medium and formula are incorrect, then fungus breeding is not good enough causes that sliding sword belongs to Fungal-feeding nematode and can not breed by normal growth；The condition such as cultivation temperature, illumination is incorrect, then fungus yellowish, poor growth, easily old and feeble, and nematicide not easily takes food；The opportunity that nematicide is inoculated in culture medium is incorrect, then affect quantity and the quality of nematicide food source；Inventor's kind to fungus, the type of culture medium and formula and condition of culture etc. have carried out a series of groping.Defining method is as follows,
Modified culture medium prescription is: in every 1000ml improved culture medium, Rhizoma Solani tuber osi 120-150g, glucose 10-12g, fishbone powder 10-12g, conch meal 6-8 part, bean cake 3-5 part, lignin 3-5 part, molasses 2-3 part, disodium hydrogen phosphate 0.01-0.02 part, potassium chloride 0.01-0.02, ferrous sulfate heptahydrate 0.01-0.02 part, all the other are water, and pH value is natural.After culture medium prepares, 121 DEG C of sterilizing 30min in high-pressure sterilizing pot.After culture medium cools down, being poured into the culture dish that diameter is 9cm, thickness is 0.7-1.0cm.By fungus Botrytis cinerea (BotrytiscinereaPers.) it is inoculated in modified culture medium, adopts sealed membrane (Parafilm) to be sealed by culture dish, to prevent the evaporation of culture medium moisture and to pollute, then culture dish is inverted and is put in constant incubator, 20-25 DEG C of dark culturing, after one week, fungus amount reproduction.Now, sliding sword being belonged to nematicide and accesses in the culture dish covering with uniformity Botrytis cinerea, inoculation method is, inoculates 50-100 μ l sterilized water with suction pipe and includes about 200 Fungal-feeding nematode through surface sterilization in media surface, sterilized water.Culture dish adopt sealed membrane (Parafilm) seal, to prevent the evaporation of culture medium moisture and to pollute.Above procedure all carries out on aseptic operating platform.Finally, being faced up by culture dish and put into the sterilizing box of suitable size, be subsequently placed in incubator, 20-25 DEG C of dark culturing, after three weeks, Botrytis cinerea disappears substantially, nematicide amount reproduction.The sliding sword of each culture dish belongs to Fungal-feeding nematode quantity and is about 19000, and breeding potential is about 95 times, and individuality is about 0.77mm, and body length/maximum body breadth is about 35.1, and it is 11.3 μm that lancet is about.
Choosing the culture medium of a wired worm of little block length, put into the modified culture medium covering with uniformity Botrytis cinerea, cunning sword is belonged to Fungal-feeding nematode and carries out expanding propagation, the sliding sword so repeating to obtain a large amount of pure culture belongs to Fungal-feeding nematode.
5, slide sword and belong to the succeeding preservation of Fungal-feeding nematode: the reproduction speed of nematicide is very fast, within every 2 weeks, produce a generation, consuming substantial amounts of fungal mycelia and spore in the process, the nutrient of culture medium and moisture also have certain time limit in addition, thus the holding time of nematicide be also a difficult point.Store method involved in the present invention is as follows.Adopting sealed membrane (Parafilm) to be sealed by culture dish, then being faced up by culture dish, it is standby to be saved in 4 DEG C of refrigerators.Every 5-6 month, sliding sword is belonged to Fungal-feeding nematode and carries out successive transfer culture, specific operation process is, culture dish is taken out from 4 DEG C of refrigerators, chooses the culture medium of a wired worm of little block length, put in the modified culture medium covering with uniformity Botrytis cinerea, face up after culture dish is sealed and put into sizeable sterilizing box, 25 DEG C of dark culturing, and whether routine observation culture dish be subject to the pollution of other funguses or acarid, is preserved as in 4 DEG C of refrigerators by culture dish after three weeks.
6, sliding sword and belong to the application of Fungal-feeding nematode: taken out from 4 DEG C of refrigerators by culture dish, culture medium in culture dish scraped with the Cornu Bovis seu Bubali spoon of sterilization, put into screen tray, the sliding sword of the separation and a large amount of pure cultures of acquisition of surface sterilization step that are sequentially carried out nematicide belongs to Fungal-feeding nematode.
Cunning sword belonging to Fungal-feeding nematode and inoculates into As polluted soil with AMF simultaneously, the sliding sword of research belongs to Fungal-feeding nematode and the interaction of AMF and the impact on tobacco growing.
It is shown that after double; two inoculations, sliding sword belongs to Fungal-feeding nematode quantity and adds 1110%, and the hyphae length of AMF improves 25%, and Mycorrhizal Infection Incidence also has a number of raising；Fungal-feeding nematode is belonged to compared with the process of AMF comparison with not inoculating sliding sword, the interaction that sliding sword belongs to Fungal-feeding nematode and AMF significantly improves the Biomass (under ground portion dry weight improves 40%, and aerial parts dry weight improves 66%) of Nicotiana tabacum L. in As polluted soil and internal arsenic cumulant (aerial parts arsenic cumulant improves 16%).Illustrate that sliding sword belongs to Fungal-feeding nematode and has superiority in As contaminated soil, promote the growth of plant in contaminated soil, when being applied in the reparation of contaminated soil, then can improve phytoremediation efficiency.
Claims (6)
1. the method that the sliding sword of enrichment culture belongs to Fungal-feeding nematode, it is characterised in that specifically comprise the following steps that
(1) slide sword and belong to the separation of Fungal-feeding nematode: from field acquisition fresh soil, adopt the tray method improved to separate nematicide from soil；
(2) slide sword and belong to the qualification of Fungal-feeding nematode: belong to the feature of Fungal-feeding nematode according to sliding sword, under anatomical lens, choose this genus nematicide with capillary tube, then it is carried out under an optical microscope identification of living body；
(3) slide sword and belong to the surface sterilization of Fungal-feeding nematode: nematicide is proceeded in the centrifuge tube of sterilizing, with streptomycin sulfate and cycloheximide mixed liquor, nematicide is carried out surface sterilization；
(4) slide sword and belong to the enrichment culture of Fungal-feeding nematode: by fungus Botrytis cinerea (BotrytiscinereaPers.) it is inoculated in sterilized modified culture medium, adopts sealed membrane to be sealed by culture dish, then culture dish is inverted and is put in constant incubator, 20-25 DEG C of dark culturing；After one week, when fungus amount reproduction, it is about to sliding sword belongs in the culture dish that uniformity Botrytis cinerea is covered with in nematicide access；Culture dish is placed in sterilizing box after sealing with sealed membrane, 20-25 DEG C of dark culturing, routine observation, until ash spore disappears substantially, and nematicide amount reproduction；Repeat the above step substantial amounts of sliding sword of acquisition and belong to Fungal-feeding nematode；Wherein, the formula of improved culture medium is: in every 1000ml improved culture medium, Rhizoma Solani tuber osi 120-150g, glucose 10-12g, fishbone powder 10-12g, conch meal 6-8 part, bean cake 3-5 part, lignin 3-5 part, molasses 2-3 part, disodium hydrogen phosphate 0.01-0.02 part, potassium chloride 0.01-0.02, ferrous sulfate heptahydrate 0.01-0.02 part, all the other are water, and pH value is natural.
2. method according to claim 1, it is characterized in that: the tray method improved described in step (1) is specific as follows: put in supporting tray by the rustless steel screen tray of 10 orders, screen tray is placed two-layer medicated napkin, then, soil sample uniform spreading on paper, add water until submergence soil in tray；It is placed under 20-25 DEG C of temperature, dark condition again and separates；After 48-60h, collect nematicide in beaker with 400 mesh sieves；Standing after overnight, slowly draw supernatant liquid with vacuum pump, and avoid disturbance lower floor liquid as far as possible, now, lower floor's liquid collects nematicide.
3. method according to claim 1, it is characterised in that: in step (3), in described mixed liquor, the quality-volumetric concentration of streptomycin sulfate is 0.80-1.20g.L-1；Quality-the volumetric concentration of cycloheximide is 0.015-0.02g.L-1；The time that nematicide carries out surface sterilization is 15-20min.
4. method according to claim 1, it is characterized in that: step (2) being identified, sliding sword belongs to Fungal-feeding nematode, the identification mark of its foundation is as follows: 1. heat kill is after death, female worm polypide is until abdomen is curved, the male worm usual afterbody whole polypide of ventrad hook is " J " shaped, and living nematode is many in serpentine；2. head is typically somewhat hung contracting, headstock osteomalacia, and lancet is relatively thin, and base portion expands, and length is generally 10-12 μm, is usually no more than 20 μm；3. esophagus presoma is cylindrical, the prosperity of middle oesophageal bulb, oval or square circular, and middle esophagus ball clack is flourishing, and rear esophageal gland leaf is flourishing, cover the back side of intestinal；4. porus excretorius is arranged in level place after oesophageal bulb；5. female worm tail is conical, tail end form of diverse: blunt circle or carefully justify, finger-like or bifurcated or have an abdomen to dash forward, with or without wake flowfield, the form of diverse of wake flowfield；6. male worm tail conical, to outside of belly hook；Copulatory spicules are Flos Rosae Rugosae thorn shape, and Ji Ding and base beak are generally flourishing, without copulatory bursa.
5. method according to claim 1, it is characterised in that: the sterilized modified culture medium described in step (4) is by culture medium in high-pressure sterilizing pot, and at 121 DEG C of temperature, sterilizing 30min obtains.
6. method according to claim 1, it is characterised in that: the store method that the sliding sword obtained after step (4) enrichment culture belongs to Fungal-feeding nematode is as follows: be placed in culture dish, and facing up after sealing to be placed in 4 DEG C of refrigerators preserves；Every 5-6 month, cunning sword is belonged to Fungal-feeding nematode successive transfer culture；Specific operation process is, culture dish is taken out from 4 DEG C of refrigerators, choose the culture medium of a wired worm of little block length, put in the modified culture medium covering with uniformity Botrytis cinerea, sterilizing box is put into after being sealed by culture dish, 20-25 DEG C of dark culturing, and routine observation, preserved culture dish as in 4 DEG C of refrigerators after three weeks.
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