CN105821112A - Method for studying feeding preference of soil fungal-feeding nematodes - Google Patents
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Abstract
The invention belongs to the field of biotechnology and particularly discloses a method for studying the fungi feeding preference of soil fungal-feeding nematodes. The method comprises the following steps: (1) separation of AM fungal spores; (2) surface disinfection of AM fungal spores; (3) preparation of an AM fungal spore germination medium; (4) germination of AM fungal spores; (5) culture of host excised root; (6) preparation of an AM fungal spore medium; (7) double culture of AM fungal spores and host excised root; (8) observation of the fungal-feeding nematodes eating AM fungi and other fungi; and (9) establishment of a selection room. The method disclosed by the invention is simple and easy to implement, can provide direct evidence of the fungal-feeding nematodes eating AM fungi and reveal the feeding preference of the fungal-feeding nematodes for the fungi in soil, and facilitates comprehensive understanding of the soil ecological process.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of study the method that soil Fungal-feeding nematode takes food preference.
Background technology
Soil Fungal-feeding nematode can take food fungus, the nutrient held by metabolic secretion and release fungus, thus affect plant growing, different impact effects depends on the relation between they and three class funguses: 1. saprophytic fungus is taken food and can improve soil nutrient mineralization rate by Fungal-feeding nematode, thus promotes the absorption of plant；2. plant pathogenic fungi is taken food and not only acts as BIOLOGICAL CONTROL effect by Fungal-feeding nematode, additionally it is possible to discharge the N of its fixing, improves nutrient mineralization rate；3. arbuscular mycorrhiza (AM) fungus is taken food and on the one hand can reduce the plant absorption to nutrient by Fungal-feeding nematode, because the effect of AM fungus helpful plant absorption nutrient, on the other hand with the nutrient in mineralising aging tissues, thus the growth of AM fungus and/or plant can be promoted.
Unfortunately, due to AM fungus still can not pure culture, therefore have not yet to see Fungal-feeding nematode and take food the positive evidence of AM fungus.In recent years, utilize dual culture technique i.e. to cultivate host's root system and AM fungus on same flat board simultaneously, set up symbiosis association and extensively proved research AM fungal morphology structure, mycelia infection processs mechanism and the effective ways of expanding propagation microbial inoculum.The method also makes us study Fungal-feeding nematode under laboratory controlled condition to take food AM fungus and become possibility.Additionally, soil fungi is of a great variety, under conditions of food resource is abundant, Fungal-feeding nematode is liked taking food which class fungus？(Ruess L, the Zapata such as Ruess
EJG, Dighton J. Food preferences of a fungal-feeding Aphelenchoides species. Nematology,
2000,2:223 230.) devise selection room (Choice
Chamber) test, i.e. separates Room 4 respectively inoculated fungis with lucite in same culture dish, and inoculation Fungal-feeding nematode of leaving a blank at culture dish center, each culturing room period sampling measuring nematode population, takes food preference with study Fungal-feeding nematode.Utilizing the method, they have studied Fungal-feeding nematodeAphelenchoides sp.17 kinds of funguses are taken food preference, find that this nematicide is partial to take food Applying Ectomycorrhizal Fungi:Cenococcum,Hymenoscyphus,Laccaria.So, in the presence of AM fungus, Fungal-feeding nematode take food how preference is again？At present the most not yet someone provides this type of method study, and method involved in the present invention to be us study that Fungal-feeding nematode takes food preference to AM fungus and other funguses provides good technical guarantee.
Summary of the invention
It is an object of the invention to provide and a kind of study the method that soil Fungal-feeding nematode takes food preference to fungus, the method is simple and practical, and effect is notable, can be the food source of research soil Fungal-feeding nematode and takes food hobby and provide technical guarantee.
The a kind of of present invention offer studies the method that soil Fungal-feeding nematode takes food preference, specifically comprises the following steps that
1, the separation of AM fungal spore: with wet screening decantation purification AM fungal spore, picking form solid colour, the uniform spore of Cytoplasm under anatomical lens, removing spore surface foreign material a moment in 42 kHz ultrasonic water bath vibrations, distilled water rinses for several times.
2, the surface sterilization of AM fungal spore: be put on the filter paper of diameter 6 cm with micropipet picking spore, carries out labelling, and by filter paper suitable for its upper cover one diameter, thus form filter holder.The filter holder that will be equipped with spore is placed in buchner funnel, moves in superclean bench, sterilizes with disinfectant solution.Disinfectant solution formula is, 2-5% oronain T, adds 2-4 and drip polysorbas20 liquid in the mixed liquor of 0.02-0.04% streptomycin and 0.01-0.02% gentamycin.AM fungal spore surface sterilization 15-20 min, rinsed with sterile water 3-5 time, to deposit standby for 0-4 DEG C, this storage temperature is conducive to the sprouting of AM fungal spore.
3, the preparation of AM fungus spore germination culture medium: 0.8-1.0% water agar, formula is that 8-10 g agar is dissolved in 1000 ml water, 121 DEG C of sterilizing 15 min.
4, the sprouting of AM fungal spore: be seeded on water agar after AM fungal spore surface sterilization, obturages culture dish mouth with sealed membrane, is then inverted culture dish, is placed in 25-30 DEG C of light culture in constant incubator.
5, the cultivation of Host Detached root: take appropriate seed and put in culture dish, is placed on superclean bench and carries out surface sterilization 10 min with 98% concentrated sulphuric acid, more standby by sterile water wash 8-10 time.Putting into a sterilizing filter paper in another sterile petri dish, addition 2-3 drips sterilized water and moistens.With little spoon picking 30-50 grain seed uniform spreading on filter paper, cover culture dish, carry out light culture at 22-25 DEG C.When seed germination grows the root of about 2 cm, cut the root of about 1 cm, proceed to improve concussion in White fluid medium and cultivate, 70 turns/min of rotating speed.The every 15 d successive transfer culture of root segment are once.
6, the preparation of AM fungal spore culture medium: by the NaH in improvement White culture medium2PO4.2H2O changes CaHPO into4.2H2O(10.7 mg), agar solidified with 1%, pH=6.0.
7, AM fungal spore and the dual cultivation of Host Detached root: Host Detached root is chosen from White fluid medium and is placed in AM fungal spore culture medium.With card punch, the culture medium that root segment is other is punched, and take out the culture medium cut, then from water agar, the AM fungal spore sprouted is taken off with same card punch, it is transferred into the vacancy that root is other, owing to aperture is consistent, the culture medium size of shift-in with remove culture medium after the vacancy that stays just coincide.After the AM fungal spore that inoculation is sprouted, with sealed membrane, culture dish is sealed immediately, to prevent water from dividing evaporation and polluting.Then culture dish is placed in constant incubator, 25-28 DEG C of light culture.Owing to AM Fungal hyphal growth has negative geotropism, during cultivation otherwise the regular position adjusting culture dish, as faced upward or downward low for culture dish, to guide AM fungal mycelia to contact earlier with root, increase and infect probability.
8, food fungus takes food the observation of AM fungus and other funguses: after AM fungal mycelia infects host's root system about 14d, and AM fungus produces a large amount of mycelia and spore.Now, 50-100 is inoculated with suction pipe
L sterilized water, in media surface, wherein includes the Fungal-feeding nematode of exhibiting high surface sterilization.Processes for disinfecting surfaces is, uses 0.80-1.20
g L -1Streptomycin sulfate and 0.015-0.02
g L-1Cycloheximide mixed liquor nematicide is carried out surface sterilization 15-20
min.Use sealed membrane to be sealed by culture dish, to prevent the evaporation of culture medium moisture and to pollute, then culture dish is faced up and be placed in constant incubator, 25-28 DEG C of light culture about 7d.Under anatomical lens, observe nematode population, observe whether nematicide takes food AM fungal mycelia or spore under an optical microscope.Finally use tray method to extract separation Fungal-feeding nematode from culture medium, count and observe nematicide indices.Use same method to observe Fungal-feeding nematode other soil fungis are taken food.
9, the foundation of room is selected: in 12 cm culture dishs, all separate Room 4 with lucite high for 12 mm, pour culture medium according to a conventional method into, culture medium thickness is about 8-10 mm, after culture medium cooled and solidified, then culture medium is chosen with sterilizing card punch in the place of Room, culture dish center that is 4 handing-over.A Room is by side, culture dish edge double inoculation Host Detached root and AM fungal spore wherein, stay in after observing that spore infects root system under anatomical lens, various funguses are inoculated by side, culture dish edge at remaining each locellus, sealed membrane (Parafilm) is used to be sealed by culture dish, then culture dish is upside down in 25-28 DEG C of light culture about 7d in constant incubator, to allow fungus amount reproduction.Being inoculated by the Fungal-feeding nematode of pure culture into culture dish center after 7d, inoculation method is, with suction pipe inoculation 50-100 L sterilized water in media surface, wherein includes the Fungal-feeding nematode of exhibiting high surface sterilization.Face up and be placed in 25-28 DEG C of light culture about 7d in constant incubator after culture dish sealing, then took out every 24 hours, measure each room Fungal-feeding nematode quantity respectively, take food preference according to what the quantity of each room Fungal-feeding nematode judged Fungal-feeding nematode.
Accompanying drawing explanation
Fig. 1: the sliding sword in each room different sample times belongs to the percentage ratio that Fungal-feeding nematode is shared in whole culture dish.
Detailed description of the invention
For the technical scheme making those skilled in the art be more fully understood that in the application, below in conjunction with the application specific embodiment, the present invention is more clearly and completely described, it is clear that, described embodiment is only some embodiments of the present application rather than whole embodiments.Based on the embodiment in the application, the every other embodiment that those of ordinary skill in the art are obtained under not making creative work premise, all should belong to the scope of protection of the invention.
Embodiment 1
1, the separation of AM fungal spore: gather enrichment culture AM fungus in Institute of Botany greenhouse (Glomus caledonium) substrate, with wet screening decantation purification spore, picking form solid colour, the uniform spore of Cytoplasm under anatomical lens, remove spore surface foreign material a moment in 42 kHz ultrasonic water bath vibrations, distilled water rinses 5-6 time.
2, the surface sterilization of AM fungal spore: be put on the filter paper of diameter 6 cm with micropipet picking spore, carries out labelling, and by filter paper suitable for its upper cover one diameter, thus form filter holder.The filter holder that will be equipped with spore is placed in buchner funnel, moves in superclean bench, sterilizes with disinfectant solution.Disinfectant solution formula is, 2-5% oronain T, adds 2-4 and drip polysorbas20 liquid in the mixed liquor of 0.02-0.04% streptomycin and 0.01-0.02% gentamycin.AM fungal spore surface sterilization 15-20 min, rinsed with sterile water 3-5 time, to deposit standby for 0-4 DEG C, this storage temperature is conducive to the sprouting of AM fungal spore.
3, the preparation of AM fungus spore germination culture medium: 0.8-1.0% water agar, formula is that 8-10 g agar is dissolved in 1000 ml water, and 121 DEG C of sterilizing 15 min, subpackage, every culture dish (d=9 cm) injects 15 ml, standby after solidification.
4, the sprouting of AM fungal spore: be seeded on water agar with micropipet after AM fungal spore surface sterilization, 6 spores equidistantly inoculated by each culture dish, obturage culture dish mouth with sealed membrane, then culture dish it is inverted, being placed in 26-30 DEG C of light culture in constant incubator, this temperature range is conducive to the sprouting of AM fungal spore.
5, butch clover (Trifolium repenseL.) cultivation of excised root: take appropriate butch clover seed and put in culture dish, is placed on superclean bench and carries out surface sterilization 10 min with 98% concentrated sulphuric acid, more standby by sterile water wash 8-10 time.Putting into a sterilizing filter paper in another sterile petri dish, addition 2-3 drips sterilized water and moistens.With little spoon picking 30-50 grain seed uniform spreading on filter paper, cover culture dish, carry out light culture at 22-25 DEG C.When seed germination grows the root of about 2 cm, cut the root of about 1 cm, proceed to improve concussion in White fluid medium and cultivate, 70 turns/min of rotating speed.Its formula is as follows: KC1 65 mg, KNO380 mg, Ca (NO3)2·4H2O 300 mg, MgSO4·7H2O 720 mg, NaH2PO4·2H20 10.7 mg, MnCl2·4H2O 4.9 mg, KI 0.75 mg, H3BO31.5 mg, ZnSO4·H2O 1.92 mg, CuSO4 · 5H2O 1 g, Na2MoO4·2H2O 0.168 g, FeNaEDTA 4.6 mg, glycine 3 mg, thiamine hydrochloride 0.1 mg, nicotinic acid 0.5 mg, pyridoxol 0.1 mg, sucrose 20 g, distilled water 1000 ml.Before sterilizing, the pH value of culture medium is transferred to 4.9, sterilizing 15-20 min at 121 DEG C.The every 15 d successive transfer culture of root segment are once.
6, the preparation of AM fungal spore culture medium: by the NaH in improvement White culture medium2PO4.2H2O CaHPO is changed into4.2H2O(10.7 mg), agar solidified with 1%, pH=6.0.
7, AM fungal spore and the dual cultivation of butch clover excised root: butch clover excised root is chosen from White fluid medium and is placed in AM fungal spore culture medium.With the card punch of a diameter of 7 mm, the culture medium that root segment is other is punched, and take out the culture medium cut, then from water agar, the AM fungal spore sprouted is taken off with same card punch, it is transferred into the vacancy that Trifolium repense grass roots is other, owing to aperture is consistent, the culture medium size of shift-in with remove culture medium after the vacancy that stays just coincide.After the AM fungal spore that inoculation is sprouted, with sealed membrane, culture medium is sealed immediately, to prevent water from dividing evaporation and polluting.Then culture dish is placed in constant incubator, 25-28 DEG C of light culture.Owing to AM Fungal hyphal growth has negative geotropism, during cultivation otherwise the regular position adjusting culture dish, as faced upward or downward low for culture dish, to guide AM fungal mycelia to contact earlier with root, increase and infect probability.
8, sliding sword to belong to Fungal-feeding nematode and take food the observation of AM fungus: after AM fungal mycelia infects butch clover root system about 14d, AM fungus produces a large amount of mycelia and spore.Now, 50-100 is inoculated with suction pipe
L sterilized water in media surface, wherein include about 200 sliding swords through the pure culture of surface sterilization belong to Fungal-feeding nematode (AphelenchoidesSp.).Processes for disinfecting surfaces is, uses 0.80-1.20
g L -1Streptomycin sulfate and 0.015-0.02
g L-1Cycloheximide mixed liquor nematicide is carried out surface sterilization 15-20
min.Use sealed membrane to be sealed by culture dish, to prevent the evaporation of culture medium moisture and to pollute, then culture dish is faced up and be placed in constant incubator, 25-28 DEG C of light culture 15 about d.It has been observed that sliding sword belongs to Fungal-feeding nematode and takes food AM fungal mycelia under anatomical lens.Use the tray method separation and Extraction nematicide improved, under anatomical lens after counting, 60 DEG C of heat-killed nematicides of gentleness, it is fixed in 4% glutaraldehyde, observes the indices of nematicide the most under an optical microscope.Statistical result shows, it is 7500 that sliding sword belongs to Fungal-feeding nematode quantity, and breeding potential is about 37.5 times, and individuality is about 0.70 mm, and body length/maximum body breadth is about 34.9, and lancet length is about 10.3 m.
9, slide sword to belong to Fungal-feeding nematode and take food the observation of other funguses: use conventional method inoculated fungi Botrytis cinerea (Botrytis cinereaPers.), chaetomium globosum (Chaetomium globosum), crust Mucor (Micheli corticolus), then use the same sliding sword of method inoculation to belong to Fungal-feeding nematode, 22-25 DEG C of light culture 15 about d.It has been observed that sliding sword belongs to Fungal-feeding nematode and all can take food above 3 kinds of funguses under anatomical lens.Statistical result shows, the culture dish of inoculation Botrytis cinerea, and it is 7300 that sliding sword belongs to Fungal-feeding nematode quantity, and breeding potential is about 36.5 times, and individuality is about 0.75 mm, and body length/maximum body breadth is about 35.2, and lancet length is about 10.3 m；The culture dish of inoculation chaetomium globosum, it is 2300 that sliding sword belongs to Fungal-feeding nematode quantity, and breeding potential is about 11.5 times, and individuality is about 0.70 mm, and body length/maximum body breadth is about 36.2, and lancet length is about 10.2 m.The culture dish of inoculation crust Mucor, it is 6400 that sliding sword belongs to Fungal-feeding nematode quantity, and breeding potential is about 32 times, and individuality is about 0.75 mm, and body length/maximum body breadth is about 36.1, and lancet length is about 10.0 m.
10, slide sword to belong to Fungal-feeding nematode and take food the observation of preference: all separating Room 4 with lucite high for 12 mm in 12 cm culture dishs, pour culture medium according to a conventional method into, culture medium thickness is about 8-10
Mm, after culture medium cooled and solidified, then takes out culture medium with sterilizing card punch in the place of Room, culture dish center that is 4 handing-over.A Room is by side, culture dish edge double inoculation butch clover excised root and AM fungal spore wherein, after observing that spore infects root system under anatomical lens after 4d, Botrytis cinerea, chaetomium globosum, crust Mucor is inoculated respectively by side, culture dish edge at remaining 3 locellus, use sealed membrane to be sealed by culture dish, then culture dish is upside down in 25-28 DEG C of light culture in constant incubator.Inoculate into culture dish center with suction pipe inoculation 150-250 l sterilized water after 7d, wherein include about 20000 sliding swords through the pure culture of surface sterilization and belong to Fungal-feeding nematode.Face up after culture dish sealing and be placed in 25-28 DEG C of light culture about 7d in constant incubator, then took out every 24 hours, the culture medium of Room 4 is scraped respectively, extract the sliding sword of separation the most respectively and belong to Fungal-feeding nematode, measure nematode population, according to the sliding sword in each room belong to Fungal-feeding nematode quantity and dynamically change judge that what sliding sword belonged to Fungal-feeding nematode takes food preference.Each sampling date is repeated 3 times, and carries out destructive sampling, whole test co-continuous observation 7d, thus has 21 culture dishs.As it is shown in figure 1, result shows that sliding sword belongs to Fungal-feeding nematode and tends to take food Botrytis cinerea and AM fungus.
Listed above is only the optimal specific embodiment of the present invention.It is clear that the invention is not restricted to above example, it is also possible to there are many deformation.All deformation that those of ordinary skill in the art can directly derive from present disclosure or associate, are all considered as protection scope of the present invention.
Claims (4)
1. study the method that soil Fungal-feeding nematode takes food preference for one kind, it is characterized in that, described method comprises the steps: the separation of step (1) AM fungal spore, the surface sterilization of step (2) AM fungal spore, the preparation of step (3) AM fungus spore germination culture medium, the sprouting of step (4) AM fungal spore, the cultivation of step (5) Host Detached root, the preparation of step (6) AM fungal spore culture medium, step (7) AM fungal spore and the dual cultivation of Host Detached root, step (8) Fungal-feeding nematode takes food the observation of AM fungus and other funguses, step (9) selects the foundation of room.
Method the most according to claim 1, it is characterised in that described method specifically includes following steps:
The separation of step (1) AM fungal spore: with wet screening decantation purification AM fungal spore, picking form solid colour, the uniform spore of Cytoplasm under anatomical lens, removing spore surface foreign material in ultrasonic water bath vibration, distilled water rinses for several times；
The surface sterilization of step (2) AM fungal spore: be put on filter paper with micropipet picking spore, carries out labelling, and by filter paper suitable for its upper cover one diameter, thus form filter holder；The filter holder that will be equipped with spore is placed in buchner funnel, moves in superclean bench, by disinfectant solution sterilization 15-20 min, rinsed with sterile water 3-5 time, deposits standby for 0-4 DEG C；
The preparation of step (3) AM fungus spore germination culture medium: 8-10 g agar is dissolved in 1000 ml water, 121 DEG C of sterilizing 15 min, to obtain final product；
The sprouting of step (4) AM fungal spore: be seeded in the culture medium of step (3) after AM fungal spore surface sterilization, obturages culture dish mouth with sealed membrane, is then inverted culture dish, is placed in 25-30 DEG C of light culture in constant incubator；
The cultivation of step (5) Host Detached root: take appropriate seed and put in culture dish, is placed on superclean bench and carries out surface sterilization 10 min with 98% concentrated sulphuric acid, more standby by sterile water wash 8-10 time；Putting into a sterilizing filter paper in another sterile petri dish, addition 2-3 drips sterilized water and moistens, and with little spoon picking 30-50 grain butch clover seed uniform spreading on filter paper, covers culture dish, carries out light culture at 22-25 DEG C；When seed germination grows the root of about 2 cm, cut the root of about 1 cm, proceed to improve concussion in White fluid medium and cultivate；
The preparation of step (6) AM fungal spore culture medium: by the NaH in improvement White fluid medium2PO4.2H2O replaces with CaHPO4.2H2O, agar solidified with 1%, pH=6.0；
Step (7) AM fungal spore and the dual cultivation of Host Detached root: Host Detached root is chosen the AM fungal spore culture medium being placed in step (6) from improvement White fluid medium；With card punch, culture medium other for root is taken out, from AM fungus spore germination culture medium, then take off the AM fungal spore sprouted with same card punch, be transferred into the vacancy that root is other；Then it is placed in constant incubator, 25-28 DEG C of light culture；
Step (8) Fungal-feeding nematode takes food the observation of AM fungus and other funguses: after AM fungal mycelia infects host root system 14d, and the sterilized water of the Fungal-feeding nematode comprising exhibiting high surface sterilization is inoculated into media surface；Use sealed membrane to be sealed by culture dish, then culture dish is faced up and be placed in constant incubator, 25-28 DEG C of light culture 7d, observes nematode population under anatomical lens, observes whether nematicide takes food AM fungal mycelia or spore under an optical microscope；Finally use tray method to extract separation nematicide from culture medium, count under anatomical lens, under microscope, observe the indices of Fungal-feeding nematode；The method of described surface sterilization is: with 0.80-1.20 g L -1Streptomycin sulfate and 0.015-0.02 g L-1Cycloheximide mixed liquor nematicide is carried out surface sterilization 15-20 min；Use same method to observe Fungal-feeding nematode other soil fungis are taken food；
Step (9) selects the foundation of room: all separates Room 4 with lucite high for 12 mm in 12 cm culture dishs, pours culture medium into, and after culture medium cooled and solidified, culture medium is chosen by the place sterilizing card punch in the handing-over of Room, culture dish center that is 4；A Room is by side, culture dish edge double inoculation Host Detached root and AM fungal spore wherein, stay in after observing that spore infects root system under anatomical lens, other soil fungis are inoculated by side, culture dish edge at remaining each locellus, use sealed membrane to be sealed by culture dish, then culture dish is upside down in 25-28 DEG C of light culture in constant incubator；After 7d, the Fungal-feeding nematode of pure culture is inoculated into culture dish center, face up after culture dish sealing and be placed in 25-28 DEG C of light culture 7d in constant incubator, then took out every 24 hours, measuring each room Fungal-feeding nematode quantity respectively, quantity and dynamic change according to each room Fungal-feeding nematode judge that Fungal-feeding nematode takes food preference to fungus.
Method the most according to claim 1, it is characterised in that:
Disinfectant solution formula in described step (2) is: at 2-5% oronain T, add 2-4 and drip polysorbas20 in the mixed liquor of 0.02-0.04% streptomycin and 0.01-0.02% gentamycin.
Method the most according to claim 1, it is characterised in that: the formula of described improvement White fluid medium is: KC1 65 mg, KNO380 mg, Ca (NO3)2·4H2O 300 mg, MgSO4·7H2O 720 mg, NaH2PO4·2H20 10.7 mg, MnCl2·4H2O 4.9 mg, KI 0.75 mg, H3BO31.5 mg, ZnSO4·H2O 1.92 mg, CuSO4 · 5H2O 1 g, Na2MoO4·2H2O 0.168 g, FeNaEDTA 4.6 mg, glycine 3 mg, thiamine hydrochloride 0.1 mg, nicotinic acid 0.5 mg, pyridoxol 0.1 mg, sucrose 20 g, distilled water 1000 ml.
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| CN107557304A (en) * | 2017-09-21 | 2018-01-09 | 河北工程大学 | A kind of AM fungies rapid propagation method |
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