CN105875200A - Method for inoculating Fungi - Google Patents
Method for inoculating Fungi Download PDFInfo
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- CN105875200A CN105875200A CN201610326970.XA CN201610326970A CN105875200A CN 105875200 A CN105875200 A CN 105875200A CN 201610326970 A CN201610326970 A CN 201610326970A CN 105875200 A CN105875200 A CN 105875200A
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- inoculation
- tool
- culture medium
- appliance
- inoculum
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- 238000000034 method Methods 0.000 title claims abstract description 70
- 241000233866 Fungi Species 0.000 title claims abstract description 15
- 238000011081 inoculation Methods 0.000 claims abstract description 98
- 239000002054 inoculum Substances 0.000 claims abstract description 40
- 239000001963 growth medium Substances 0.000 claims abstract description 26
- 235000017166 Bambusa arundinacea Nutrition 0.000 claims abstract description 24
- 235000017491 Bambusa tulda Nutrition 0.000 claims abstract description 24
- 241001330002 Bambuseae Species 0.000 claims abstract description 24
- 235000015334 Phyllostachys viridis Nutrition 0.000 claims abstract description 24
- 239000011425 bamboo Substances 0.000 claims abstract description 24
- 229910052751 metal Inorganic materials 0.000 claims abstract description 20
- 239000002184 metal Substances 0.000 claims abstract description 20
- 230000002538 fungal effect Effects 0.000 claims description 17
- 241000894006 Bacteria Species 0.000 claims description 11
- 235000001727 glucose Nutrition 0.000 claims description 5
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- 239000000463 material Substances 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 229910000831 Steel Inorganic materials 0.000 claims description 4
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- 239000007787 solid Substances 0.000 claims description 3
- 230000000844 anti-bacterial effect Effects 0.000 claims description 2
- 230000015572 biosynthetic process Effects 0.000 claims description 2
- 150000002304 glucoses Chemical class 0.000 claims description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 2
- 239000010931 gold Substances 0.000 claims description 2
- 229910052737 gold Inorganic materials 0.000 claims description 2
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- 244000052769 pathogen Species 0.000 description 23
- 230000001717 pathogenic effect Effects 0.000 description 23
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- 241001477873 Cornus sanguinea Species 0.000 description 9
- 201000010099 disease Diseases 0.000 description 6
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- 235000013312 flour Nutrition 0.000 description 1
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- 235000013305 food Nutrition 0.000 description 1
- 239000000446 fuel Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000002655 kraft paper Substances 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 238000002663 nebulization Methods 0.000 description 1
- 230000024241 parasitism Effects 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
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- 239000011691 vitamin B1 Substances 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Mycology (AREA)
- Environmental Sciences (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses a method for inoculating fungi, an inoculation appliance and a culture medium. The inoculation appliance and the culture medium are applied to the method. The method for inoculating the fungi includes sterilizing the metal inoculation appliance; transferring the inoculation appliance to the culture medium with the target fungi in a sterile environment and then cultivating the culture medium; contaminating inoculums formed by wood or bamboo components on the inoculation appliance with the fungi; taking out the inoculation appliance and piecing an inoculation position of a host by the aid of a tip of the inoculation appliance; infecting the host with the fungi on the contaminated wood or bamboo inoculums on the inoculation appliance to finally implement inoculation operation. The tip is arranged at an end of the metal inoculation appliance, and the wood or bamboo components are arranged on the inoculation appliance. The method, the inoculation appliance and the culture medium have the advantages of accuracy, convenience, capability of saving time and labor and the like. Besides, the fungi can be inoculated on hard portions of woods and the like by the aid of the method, the inoculation appliance and the culture medium.
Description
Technical field
The present invention relates to a kind of fungal inoculum method, and the inoculation utensil that used of this method and culture medium, definitely
Say it is a kind of to utilize inoculating tool to make targeted fungal contact with the inoculation portion of host plant, make fungus invade the inoculation of host plant
Method and inoculation utensil and culture medium.
Background technology
Manually make pathogenic fungi and the susceptible location contacts of host plant, create conditions and make pathogen invade and lead to host to send out
Sick process is inoculation.Inoculation is the important step of the sick process of card, at research parasitism occurrence regularity, measures Variety Disease-resistance
Property, medicament protection effect is required for inoculation when evaluating.Therefore, inoculation method directly affects test effect.
Plant disease Inoculation Method, is the spread path according to disease and infection way design, plant disease
Kind is a lot, its spread path and infection way different.The inoculation method of fungus has the method without inoculating tool and adopting of can using
By method two class of inoculating tool, the method for inoculating tool is wherein used to have:
1) nebulization, this method is the spore suspension that will have configured, and is sprayed on host surfaces, and pathogen can be from pore, wound
Mouth or epidermis invade.
2) semar technique, pathogen brush pen or brushing will be put on plant surface, make spore germination invade.
3) injection, is injected into spore suspension syringe and remembers growing point or young tender part.
4) needle point method, after artificial manufacture mechanical damage, is attached to wound induction disease by bacterium cake.
Existing inoculation method can be found in " planting disease research method " Fang Zhongda, and 1998.But traditional inoculation method often has
The shortcomings such as having inoculation efficiency low, symptom occurs slow, inoculation concordance difference, and in aforesaid method, the method for 1 and 2 is after inoculation
Also need to moisturizing 48 hours, and the less individuality for plantlet stage of method 3 and 4, and the organizational structure such as excised leaf, very
Difficult quantitatively enforcement is infected.
In recent years, microbionation method is improved many, and fungal inoculum method almost has no change.
Chinese invention patent 201410604178.7 discloses the manufacture method of a kind of edible fungi peg wood strain, belongs to edible
Bacterium produces field of planting.The method comprises the steps: to order gauze or kraft paper to put in one end of peg wood in lime water soaks
Steep to peg wood without the white heart, pull peg wood out and dry table water；By flour, Semen Maydis powder, white sugar, Gypsum Fibrosum, potassium dihydrogen phosphate, sulphuric acid
Magnesium, mushroom Feng Su, vitamin B1It is added in water, is heated to starchiness and obtains binding agent；Mix to glue by peg wood and binding agent
Mixture is uniformly attached to peg wood surface, when adding wheat bran or Testa oryzae stirring to its uniform adhesion peg wood surface 70-80%, by peg wood
Load in Polypropylene Bag or seed bottle, sterilizing after sealing；Aseptically inoculate strain, be placed in 20-26 DEG C and cultivate 10-20
My god, mycelia can be covered with.But this peg wood strain is not suitable for fungal inoculum, and its preparation method is the most complicated.
A kind of open wooden peg strain method of Chinese invention patent application 201310720075.2, the inoculation wood used by this patent
Nail is to be fabricated to, with branch, waste wood, substandard products food label rod, fritters of twisted dough, Fructus Pruni bar, the cuspidated bar-shaped wooden peg of band that rugosity is 0.5cm, its
During use be soak with the room temperature white sugar water of 5% concentration within 24 hours, pull out admix again bacterium rivet nut long-pending 10% fuel can pack
For inoculating.But this wooden peg immerses culture medium can be softened, then with this wooden peg the harder host of quality, such as on trees,
Host may be penetrated into, it is achieved inoculation operation during inoculation.
Summary of the invention
The present invention provides a kind of fungal inoculum method overcoming prior art not enough, is given used by this method simultaneously
Culture medium and inoculation utensil.
The fungal inoculum method of the present invention is to use inoculating tool to make targeted fungal contact with the inoculation position of host equally,
Making fungus invade host, its concrete way is: first will its one end metal be tip, on it with wooden or bamboo matter portion
The inoculating tool sterilizing of part, then after having the culture medium of targeted fungal in an aseptic environment aforesaid inoculating tool being transferred to it
Culture medium is cultivated, more right after having the culture medium of targeted fungal in an aseptic environment aforesaid inoculation tool being transferred to it
Culture medium is cultivated, and makes inoculation tool upper wooden or the inoculum microbiological contamination of bamboo matter parts composition, then takes out inoculation tool and by it
The inoculation position of host is thrust at tip, makes the wooden or bamboo matter inoculum fungal infection host of microbiological contamination on inoculation tool, it is achieved
Inoculation operation.
The culture medium that fungal inoculum method of the present invention uses is added with 20 grams of glucoses in 1L Rhizoma Solani tuber osi decocting liquid.
The inoculation tool that the inoculation method of the present invention uses is at least by carrying cuspidated bar-shaped metalwork and being embedded in bar-shaped gold
Belong to the wooden or bamboo matter on part or other material that antibacterial, fungus can be made to adhere to thereon constitutes formation inoculum, Qi Zhongjie
Plant at least part of outer surface exposing to bar-shaped metalwork of body.
The inoculation tool that the inoculation method of the present invention uses can also is that by wooden or bamboo its one end band cone, cone
After be set to cylindrical inoculum and be fixed on cone head metal tip constitute.
Preferably, inlay fixing between the metal tip of the inoculation tool of the present invention and wooden or bamboo cone, or use spiral shell
Stricture of vagina is fixed.
The inoculation tool of the present invention, it is characterised in that the metal used by inoculation tool is rustless steel.
The invention have the advantage that
1, stab, especially owing to the present invention uses band cuspidated metal club shaped structure, particularly rustless steel needle point to be easy to tissue
It is suitable for the forest inoculation that trunk is hard, is completely absent in prior art the deficiency that inoculation wooden peg softens.On the other hand, originally
As long as the inoculation tool of invention cleans after a procedure, the most reusable after sterilizing, it is possible to inoculum will be changed under special circumstances,
Therefore the inoculation tool escapable cost of the present invention is used.
2, the upper wooden or inoculum of bamboo matter arranged of inoculation tool of the present invention, i.e. can be with microbiological contamination to infect host, simultaneously
It is more convenient for accurate surveying pathogen p of E, accurate ocean weather station observation and statistics.
3, time-consuming.The method of the present invention is the same with additive method, needs pathogen pure culture, but was all solid in the past
Culture medium culturing, waits that pathogen produces spore.Some produces spore comparatively fast such as rod method, but a lot of pathogen product spore cycle is long, sporulation quantity
Little, need configuration to reach 1 × 106The spore suspension of individual/ml concentration, generally requires the pathogen of a large amount of expanding propagation, incubation time
At 20-60 days.In the method for the present invention, inoculum is cultivated in liquid base, growth of pathogenic bacteria can be made rapid and abundant
Microbiological contamination, this process only needs 10-15 days time.
4, error is little.Prepared spore suspension in the past or spore was smeared, in order to ensure to reach higher pathogen concentration,
Need to prepare spore suspension with blood counting chamber.But blood counting chamber statistical data is only the statistics of a multiple conversion
Estimated value, often with practical situation error relatively greatly (such as: reach 1 × 10 with blood counting chamber statistics spore concentration6Individual/ml, and real
Border may only have 1 × 105Individual/ml, multipotency differ from 10 times).The method of the present invention need not prepare spore suspension, energy on shaking table
Ensure each inoculum institute content of molds uniformity, it is to avoid produce error.
5, uniformity is good.Traditional spore suspension is inoculated, is smeared the methods such as inoculation, is simply connect by certain density spore
Kind to plant, but uniformity it is difficult to ensure that, especially inoculate the less individuality such as seed, seedling, it may occur however that the phenomenon missed kind
(such as spray inoculation method: some the surface of the seed may touch 10 spores, and some possible 1 does not all have in spraying).This
The method of invention is in inoculating tool preparation process, and at the uniform velocity shaking table makes inoculation tool upper wooden or bamboo matter inoculum is abundant with pathogen
Mixing, ensure that the consistent of each inoculation tool content of molds from maximum of probability, thrusts plant, it is ensured that inoculated in seeded process
In journey, pathogen quantity is uniform, consistent with between process.
6, rapid onset.The methods such as spray inoculation method, smear inoculation, need to wait for pathogen and sprout, and germ tube grows, then is formed
Attachment spore, invaded plants, can be affected by plant epidermis wax coat, horny layer etc., occur when symptom generally requires about 10 days
Between.The method of the present invention is the needle point method inoculation improved, and directly contributes wound, can make rapidly pathogen invaded plants, about 3-4
It i.e. can be observed symptom, investigates the harm that plant is caused by pathogen, measures pathogen growth in plant, extension speed
Degree.
7, fixed point property is strong.Inoculation method in the past, is not easy to from macroscopically determining it is which spore germination intrusion is planted actually
Thing, is easily subject to interacting of many places p of E during concrete observation.And the method for the present invention, p of E is fixed, i.e. from acupuncture
Place starts to infect, the easily observation process of pathogen invaded plants, the degree, and one-point measurement pathogen extension speed on plant
Degree.
8, the present invention inoculation tool the most practical and can make more convenient to operate simply.After conventional needle acupuncture manipulation, also use spore
Suspension or pure culture biscuits involvng inoculation.Spore suspension still suffers from above-mentioned shortcoming.And pure culture biscuits involvng inoculation method there is problems in that 1 bacterium colony
The bacterium cake content of molds that zones of different produces is different, and pathogen activity is different, so concordance is poor between different bacterium cake processes；2 bacterium cakes
Seeded process needs other instruments auxiliary to manufacture wound, and after pure culture biscuits involvng inoculation, moisturizing to be closed, easily pollutes；Wooden inoculating tool
Easily occur to soften and be difficult to penetrate into harder host.The tip that inoculation tool front end is a metal of the present invention, inherently manufactures
The instrument of wound；Add is to prepare with metal, due to the reason of texture material so that it is have suitable rigidity easily to penetrate into heavily fortified point
In hard wooden host, when with culture medium effect, do not affect the rigidity of its metal tip when inoculum simultaneously, more do not result in
The deficiency of existing wooden peg front end deliquescing；Wooden or the bamboo matter parts of the embedding in inoculation tool itself form an inoculum, it
Can be with pathogen co-cultivation, a large amount of pathogen of portability, make inoculation operation simple and convenient.Institute with the inventive method and connects
Plant tool and gathered multiple action, and operation can be made the simplest.
9, used medium cost is lower.Culture medium used by the present invention is the Rhizoma Solani tuber osi decocting liquid Rhizoma Solani tuber osi added with glucose
Decocting liquid.And the fungi culture medium now used is glucose agar medium (PDA), although and the method for the present invention is still one
PDA culture medium, but wherein need not add agar, therefore its cost prepared relatively prior art is lower.
Accompanying drawing explanation
Accompanying drawing 1 is the inoculation tool schematic diagram of the present invention.In figure: 1 is the bar-shaped metalwork of inoculation tool, and 2 is bar-shaped for being embedded in
Wooden or the parts of bamboo matter on metalwork, 3 is the tip portion of bar-shaped metalwork.
Accompanying drawing 2 is that the A-A of accompanying drawing 1 is to generalized section.
Accompanying drawing 3 inoculates another embodiment schematic diagram of tool for the present invention.
Accompanying drawing 4 is the postvaccinal photo of the present invention.
Accompanying drawing 5 is to use the method inoculation of the present invention and nonvaccinated contrast photo.
Detailed description of the invention
The following is embodiments of the invention.
One embodiment of the inoculation tool of the present invention is as it is shown in figure 1, be by bar-shaped metalwork 1 and to be embedded in bar-shaped metalwork
On wooden or bamboo matter inoculum 2 constitute, the front end of bar-shaped metalwork 1 is a tip portion.Reality prepare in permissible
On bar-shaped metalwork 1, offer a through hole as shown in Figure 1, then in through hole, inlay one piece of connecing by wooden or bamboo material
Planting body 2, the two ends of such inoculum 2 can expose to contact with cultivation bacterium.Inoculation tool can also be formed in bar-shaped metalwork 1
On offer a blind hole, inlay in blind hole one piece by the wooden or inoculum of bamboo material, in this structure, inoculum will only
There is one to be exposed, one jackscrew can be set on top, the hole of blind hole, rotate jackscrew and can will inlay the inoculum into blind hole
Eject blind hole so that changing.
Implementing such as Fig. 3 and show for the 3rd of the inoculation tool of the present invention, it is by wooden or bamboo its one end band cone, circle
It is set to cylinder after cone, and inlays the metal tip composition in cone head.In the inoculation tool of the present invention, its metal tip
Inlay fixing between end and wooden or bamboo cone, or with being screwed, i.e. at metal tip and wooden or bamboo circle
Cone each arranges the female thread of the pin thread cooperated with the metal metal pointing end place of matching.
The metal used by inoculation tool of the present invention is preferably with rustless steel.
The size of one actual product of the inoculation tool of the present invention is as follows: the long 10mm of its cylindrical portion, diameter 2mm, point
The long 5mm of end portion.
The inoculation method of the present invention is as follows:
1, the preparation of culture medium
200g Rhizoma Solani tuber osi is cut into small pieces, adds water and well-done (boil 20 ~ 30 minutes, can be poked by Glass rod, use filtered through gauze
Putting in pot, add water 1000ml, then adds glucose 20g, then 121 DEG C of autoclaving 30min wherein, then is divided in
250ml triangular flask saves backup.
2, pathogenic fungi is cultivated
By target pathogenic bacteria after purification, get 5mm diameter bacterium cake, add in the fluid medium in aforesaid triangular flask, put
On constant-temperature table (adjusting temperature according to various pathogenic bacteria), rotating speed 150r/min, cultivates 10-15 days, pathogen continued propagation.
3, the preparation of inoculation tool
Inoculation is had 121 DEG C of high pressure 30min sterilizings 2 times, immerses in potato dextrose broth after sterilizing for the 3rd time, then
Transfer to inoculation tool in an aseptic environment, in the pathogen triangular flask of liquid culture, every 250ml add tens of pieces of inoculation tools.
Then proceed to be placed on constant-temperature table (adjusting temperature according to various pathogenic bacteria) and carry out Bacteria culturing, shaking speed 150r/
Min, incubation time about 72h.
4, inoculation
From culture medium, take out the inoculation tool of microbiological contamination, utilize the tip of its front portion to thrust the children being defined as host in an aseptic environment
Shoot root stem, or the position such as blade or trees stem, cause artificial wound, make inoculum part connect with host's inoculation position
Touching, can promote that pathogen invades from wound, infect for excised leaf, disease system infects etc. is tested.See accompanying drawing 4.
Accompanying drawing 5 is to use the method inoculation of the present invention and nonvaccinated contrast photo.From being positioned at photo bottom seen from accompanying drawing 5
Host be vaccinated infection, inoculation position has presented infection phenomenon, although and the plant on photo top is also stabbed wound, but
The most do not present infection phenomenon.
For checking the reliability of inoculum, 10 inoculation tools are placed in sterilizing culture dish (90mm), add 5ml aseptic
Water, fully agitation shake up, and wash the Sporulation suspension on lower inoculum surface.Under simple microscope, (100 ×) add up 20
Spore quantity in the visual field, calculates the spore quantity on each inoculum according to microscopical visual field diameter and culture dish internal diameter.
Totally 10 repetitions.Record with eyepiece micrometer, microscope 100 × under each visual field a diameter of 2mm, 90mm culture dish bottom in
Footpath is 85mm, and culture dish bottom has 1805.25 visuals field, and each inoculum band spore count is:
.Each inoculum institute band spore count is 48～62, average out to 56, through SPSS variance analysis, each inoculum institute band spore
Quantum count is without significant difference.
By above content, inoculation this method of the present invention has the features such as accurate, convenient.Can for forest etc. relatively
Hard part inoculation, can directly order into plant by inoculum, time saving and energy saving.
Claims (7)
1. a fungal inoculum method, uses inoculating tool to make targeted fungal contact with the inoculation position of host, makes fungus invade
Host, it is characterised in that: first its one end metal is tip, goes out with wooden or bamboo matter parts inoculating tools on it
Bacterium, then culture medium is trained after having the culture medium of targeted fungal in an aseptic environment aforesaid inoculating tool being transferred to it
Support, then take out inoculating tool and host's inoculation position is thrust at the tip one of inoculating tool.
2. the culture medium that a kind of fungal inoculum method described in claim 1 uses, it is characterised in that culture medium used be
Added with 20 grams of glucoses in 1L Rhizoma Solani tuber osi decocting liquid.
3. its one end that a kind of fungal inoculum method described in claim 1 uses is with most advanced and sophisticated inoculation tool, it is characterised in that
Described inoculation tool at least by the wooden or bamboo matter carrying cuspidated bar-shaped metalwork and be embedded on bar-shaped metalwork or other
Antibacterial, fungus can be made to constitute formation inoculum composition at the material of upper attachment, and wherein inoculum is at least part of exposes to
The outer surface of bar-shaped metalwork.
4. one end band cuspidated inoculation tool that a kind of fungal inoculum method described in claim 1 uses, it is characterised in that be
It is set to cylindrical inoculum by after wooden or bamboo its one end band cone, cone and is fixed on the gold of cone head
Belong to tip to constitute.
Inoculation tool the most according to claim 4, it is characterised in that inlay solid between metal tip and wooden or bamboo cone
Fixed.
6. have according to the inoculation described in claim 4, it is characterised in that solid with screw thread between metal tip and wooden or bamboo cone
Fixed.
7. have according to the inoculation described in claim 3 or 5 or 6, it is characterised in that the metal used by inoculation tool is rustless steel.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610326970.XA CN105875200A (en) | 2016-05-17 | 2016-05-17 | Method for inoculating Fungi |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610326970.XA CN105875200A (en) | 2016-05-17 | 2016-05-17 | Method for inoculating Fungi |
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| Publication Number | Publication Date |
|---|---|
| CN105875200A true CN105875200A (en) | 2016-08-24 |
Family
ID=56716333
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|---|---|---|---|
| CN201610326970.XA Pending CN105875200A (en) | 2016-05-17 | 2016-05-17 | Method for inoculating Fungi |
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN2759178Y (en) * | 2004-09-23 | 2006-02-22 | 崔普铉 | Support for fishing rod |
| CN201138955Y (en) * | 2007-12-31 | 2008-10-29 | 郭宏旗 | Glossy ganoderma mushroom deep layer positioning beads inoculator |
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