CN108865896A - A kind of method of Fusarium oxysporum Cuba specialized form bacterial strain rejuvenation - Google Patents
A kind of method of Fusarium oxysporum Cuba specialized form bacterial strain rejuvenation Download PDFInfo
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- CN108865896A CN108865896A CN201810623456.1A CN201810623456A CN108865896A CN 108865896 A CN108865896 A CN 108865896A CN 201810623456 A CN201810623456 A CN 201810623456A CN 108865896 A CN108865896 A CN 108865896A
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- bacterial strain
- rejuvenation
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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Abstract
The invention discloses a kind of methods of Fusarium oxysporum Cuba specialized form bacterial strain rejuvenation, by aseptically by pathogenicity decline Foc bacterial strain tieback to sterile water planting rooting tube plantlet, then the separation of pathogen is aseptically carried out, it not only fundamentally solves the problems, such as living contaminants, but also eliminates the link of surface sterilization；Switching purifying is carried out without to bacterium colony after being separately cultured, the preparation of spore suspension is can be directly used for, simplifies operating procedure.It can be quickly obtained rejuvenation Foc bacterial strain using method provided by the invention, push the process of banana blight breeding for disease resistance work.
Description
Technical field
The invention belongs to agricultural-microorganisms technical fields, and in particular to a kind of Fusarium oxysporum Cuba specialized form bacterial strain is multiple
Strong method.
Background technique
Research of fusarium wilt disesase of banana is by Fusarium oxysporum Cuba specialized form Fusarium oxysporum
F.sp.cubense (Foc) infects silborne fungal diseases caused by banana root.Currently, the disease spread to the whole world it is each main
Banana production state becomes one of most destructive banana disease.For a long time, to different banana strains or belong to kind according to Foc
Difference in Pathogenicity be divided into 4 biological strains.Wherein No. 4 biological strains (race 4) is pathogenic most strong, can not only
All cultivars susceptible to No. 1 biological strain and No. 2 biological strains are infected, the main cultivation of China's banana production can be also infected
Train kind --- fragrant tooth any of several broadleaf plants (Musa AAA Cavendish subgroup).
Many decades have been carried out in the research of banana blight prevention and control field, can be effectively controlled this under field conditions at present
Disease biology, chemical prevention means and cultivation step it is still extremely limited；For the plot fallen ill, planting disease-resistant variety is
The only effective prevention and control measure.In recent years, it is obtained by the somaclonal variation generated in the quick reproductive process of Banana In Vitro
Obtained a series of disease-resistant strains.But generally speaking, the quantity of existing disease-resistant variety is still extremely limited；On the other hand, part is anti-
There are also to be further improved for the economical character of sick kind.
The weight that Disease Resistance Identification is the work of banana blight breeding for disease resistance is carried out by No. 4 biological strains of artificial infection Foc
Want link.But the pathogenicity that in actual application, squamous subculture frequently results in bacterial strain morphs, therefore bacterial strain is kept to cause
Sick power and to pathogenicity decline bacterial strain carry out rejuvenation be very necessary.Currently, carrying out tieback rejuvenation to plant pathogenic fungi
During, for the interference for excluding miscellaneous bacteria, surface sterilization must be carried out to vegetable material when separating pathogen after tieback again, to
Bacterium colony will also carry out switching purifying to it after growing, to ensure no varied bacteria growing.So far there is not been reported have it is larger it is improved its
Its rejuvenation method.
Summary of the invention
It is an object of the present invention to provide a kind of methods of Fusarium oxysporum Cuba specialized form bacterial strain rejuvenation.
The technical solution adopted by the present invention is that：
A kind of method of Fusarium oxysporum Cuba specialized form bacterial strain rejuvenation, includes the following steps：
1) by pathogenicity decline bacterial strain tieback to sterile water planting rooting tube plantlet.Aseptically, according to 1:10~15
Ratio the spore suspension of pathogenicity decline bacterial strain is added the fluid nutrient medium of sterile water planting rooting tube plantlet and shakes up；
2) after tieback 3~4 weeks, the separation for having the rooting tube plantlet of disease symptom to carry out pathogen is chosen.In aseptic condition
Under from the rooting tube plantlet base portion of above-mentioned tool classical symptom cut bulb thin slice, be directly inoculated into culture medium culture；
3) after bacterium colony is grown, several bacteria cakes aseptically are cut along colony edge, be put into containing 0.05%~
In the sterile water of 0.10% Tween-20, shake up at a slow speed to get the spore suspension of 1 generation of rejuvenation bacterial strain is arrived, and detect spore count
Mesh；
4) aseptically, according to the method for step 1), rejuvenation 1 generation bacterial strain tieback to sterile water planting is taken root test tube
Seedling；
5) after tieback 3~4 weeks, the spore suspension of 2 generation of rejuvenation bacterial strain is arrived according to step 2)~step 3) method
Liquid；
6) aseptically, according to the method for step 1), rejuvenation 2 generation bacterial strain tieback to sterile water planting is taken root test tube
Seedling；
7) 3-4 weeks after the 3rd tieback, according to step 2)~step 3) method to get outstanding to the spore of 3 generation of rejuvenation bacterial strain
Supernatant liquid, and the pathogenicity of rejuvenation 3 generation bacterial strain is measured.
Further, final concentration of 10 of the fluid nutrient medium miospore after inoculation described in step 1)5A spore/
mL。
Further, the rooting tube plantlet of disease symptom described in step 1) is yellowing leaf or withered test tube of taking root
Seedling.
Further, bulb thin slice described in step 2) is cut from yellowing leaf or withered rooting tube plantlet base portion.
Further, culture medium described in step 2) is PDA culture medium.
Further, condition of culture described in step 2) is 25~28 DEG C of cultures 5~7 days.
Further, rooting tube plantlet base portion laterally cuts bulb thin slice in step 2).
Further, bulb sheet thickness described in step 2) is about 1~3mm.
Further, bacteria cake diameter described in step 3) is 5~7mm.
Further, the bacterial strain is Fusarium oxysporum Cuba specialized form bacterial strain.
Further, the bacterial strain is No. 4 biological strain bacterial strains of Fusarium oxysporum Cuba specialized form.
Application of the method described in any of the above embodiments in bacterial strain rejuvenation.
Further, the bacterial strain is Fusarium oxysporum Cuba specialized form bacterial strain.
Further, the bacterial strain is No. 4 biological strain bacterial strains of Fusarium oxysporum Cuba specialized form.
The beneficial effects of the invention are as follows：
Since the sterile water planting rooting tube plantlet of pathogenicity decline Foc bacterial strain tieback and subsequent pathogenicbacteria separation are sterile
Under the conditions of carry out, fundamentally solve the problems, such as living contaminants；Separate pathogen when without the tissue to site of pathological change into
Row surface sterilization can directly be inoculated into PDA culture medium, carry out switching purifying without to bacterium colony after being separately cultured, can be direct
For the preparation of spore suspension, operating procedure is simplified；Method provided by the invention has preferable controllability, in addition to being used for
It has failed the quick rejuvenation of Foc bacterial strain, it may also be used for " the broad sense rejuvenation " of Foc bacterial strain occurs aobvious in the pathogenicity of Foc bacterial strain
Before writing decline, tieback is periodically carried out to it and is separately cultured, to keep the viability and pathogenicity of Foc bacterial strain.
Detailed description of the invention
Fig. 1 is the operating process schematic diagram of pathogenicity of the present invention decline Foc bacterial strain tieback rejuvenation；
Fig. 2 is the effect for carrying out rejuvenation to Fusarium oxysporum Cuba specialized form bacterial strain using method of the invention.
Specific embodiment
The present invention is further illustrated with reference to the accompanying drawing, but is not limited to embodiment.
Embodiment 1
With ' Brazil ' banana (Musa AAA Cavendish subgroup) for experimental material.' Brazil ' banana is current
One of domestic most important cultivar of banana production, the kind are susceptible to No. 4 biological strain height of Foc.According to Shoot Tip Culture
Conventional method and step Vitro Quick Reproduction is carried out to ' Brazil ' banana, and obtain rooting tube plantlet.
On this basis, referring to patent " a kind of in vitro hydroponic system of banana root system and cultural method " (publication number：
CN107646661A the hydroponic system of ' Brazil ' banana rooting tube plantlet) is established, and obtains water planting rooting tube plantlet.
For No. 4 biological strain II5 bacterial strains of Foc for occurring significantly to fail using pathogenicity as initial strains, II5 bacterial strain spore is outstanding
Supernatant liquid (106A spore/mL) preparation conventionally carry out.
Aseptically, by initial II5 bacterial strain tieback to ' Brazil ' banana water planting rooting tube plantlet.When inoculation, by 5mL
The spore suspension of II5 bacterial strain is directly added into the fluid nutrient medium of about 60mL ' Brazil ' banana water planting rooting tube plantlet and shakes up,
To ensure that the root system of disease fungus and banana plantlet in vitro comes into full contact with.After spore suspension is added, fluid nutrient medium miospore
Final concentration should reach 105A spore/mL.
3-4 weeks after 1st tieback, the incidence of ' Brazil ' banana water planting rooting tube plantlet is observed, chooses yellowing leaf
Or withered rooting tube plantlet carries out the separation of pathogen.Aseptically from above-mentioned tool classical symptom, (yellowing leaf is withered
Wither) plantlet base portion laterally cut the bulb thin slice of thickness about 2mm, be directly inoculated into PDA culture medium, 25 DEG C of culture 5-7d.
After bacterium colony is grown, the bacteria cake of diameter 6mm is aseptically cut from the colony edge of PDA plate with punch
It 5-10, is put into sterile water of the 20mL containing 0.05% Tween-20, shakes 5min at a slow speed to get the spore of 1 generation of rejuvenation II5 bacterial strain is arrived
Sub- suspension.Take a part of above-mentioned spore suspension blood cell plate counter detection spore number.
Aseptically, rejuvenation 1 generation II5 bacterial strain tieback is taken root to the sterile water planting of banana according to aforementioned same method
Test tube seedling.
3-4 weeks after 2nd tieback, the separation of pathogen is carried out according to aforementioned same method, prepares 2 generation of rejuvenation II5 bacterium
The spore suspension of strain.
Aseptically, rejuvenation 2 generation II5 bacterial strain tieback is taken root to the sterile water planting of banana according to aforementioned same method
Test tube seedling.
3-4 weeks after 3rd tieback, the separation of pathogen is carried out according to aforementioned same method, prepares 3 generation of rejuvenation II5 bacterium
The spore suspension of strain.According to patent of invention has been authorized, " disease resistance of a kind of pair of research of fusarium wilt disesase of banana carries out Rapid identification
Method " (the patent No.:ZL200910192176.0) pathogenicity of rejuvenation 3 generation II5 bacterial strain is measured.Fig. 1 is experiment stream
Cheng Tu.
Embodiment 2
No. 4 biological strain ACC31282 of Foc for occurring significantly to fail using pathogenicity is initial strains, ACC31282 bacterial strain
Spore suspension (106A spore/mL) preparation conventionally carry out.
Aseptically, by initial ACC31282 bacterial strain tieback to ' Brazil ' banana water planting rooting tube plantlet.When inoculation,
The spore suspension of 5mL ACC31282 bacterial strain is directly added into the liquid training of about 60mL ' Brazil ' banana water planting rooting tube plantlet
It supports base and shakes up, to ensure that the root system of disease fungus and banana plantlet in vitro comes into full contact with.After spore suspension is added, Liquid Culture
The final concentration of base miospore should reach 105A spore/mL.
3-4 weeks after 1st tieback, the incidence of ' Brazil ' banana water planting rooting tube plantlet is observed, chooses yellowing leaf
Or withered rooting tube plantlet carries out the separation of pathogen.Aseptically from above-mentioned tool classical symptom, (yellowing leaf is withered
Wither) plantlet base portion laterally cut the bulb thin slice of thickness about 2mm, be directly inoculated into PDA culture medium, 25 DEG C of culture 5-7d.
After bacterium colony is grown, the bacteria cake of diameter 6mm is aseptically cut from the colony edge of PDA plate with punch
It 5-10, is put into sterile water of the 20mL containing 0.05% Tween-20, shakes 5min at a slow speed to get 1 generation of rejuvenation ACC31282 bacterial strain is arrived
Spore suspension.Take a part of above-mentioned spore suspension blood cell plate counter detection spore number.
Aseptically, according to aforementioned same method by rejuvenation 1 generation ACC31282 bacterial strain tieback to banana sterile water
Train rooting tube plantlet.
3-4 weeks after 2nd tieback, the separation of pathogen is carried out according to aforementioned same method, prepares 2 generation of rejuvenation
The spore suspension of ACC31282 bacterial strain.
Aseptically, according to aforementioned same method by rejuvenation 2 generation ACC31282 bacterial strain tieback to banana sterile water
Train rooting tube plantlet.
3-4 weeks after 3rd tieback, the separation of pathogen is carried out according to aforementioned same method, prepares 3 generation of rejuvenation
The spore suspension of ACC31282 bacterial strain.
Further effect inspection is done to rejuvenation bacterial strain prepared by the present invention below.
Susceptible variety ' Brazil ' and disease-resistant variety ' agriculture section 1 ' it is inoculated with initial II5 bacterial strain and rejuvenation 3 generation II5 bacterial strain 4 respectively
Zhou Hou carries out disease level identification to single plant rooting tube plantlet according to 0-6 grades of disease opinion ratings, wherein 0 grade-plantlet
Growth is normal, without any disease symptom；The vanelets of 1 grade-vacation basal part of stem are withered, but the color of false stem itself does not become
Change；The dimmed region of 2 grades-false stem color is less than or equal to the 1/2 of entire false stem height；The dimmed region of 3 grades-false stem color
More than the 1/2 of entire false stem height；4 grades-flavescence or withered upper leaf the piece number are less than or equal to plantlet top total leaf number
1/2；5 grades-flavescence or withered upper leaf the piece number are more than the 1/2 of plantlet top total leaf number；6 grades-whole strain test tube seedling
Withered death.
The result shows that：Tieback rejuvenation carried out to II5 bacterial strain by continuous 3 times, the Mean disease grade of ' Brazil ' banana by
2.67 originally are improved to 5.13 (Fig. 2), reach the Mean disease grade of banana blight susceptible variety, it was demonstrated that rejuvenation 3 generation II5
The pathogenicity of bacterial strain is restored.The pathogenicity of rejuvenation 3 generation ACC31282 bacterial strain is also restored.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (10)
1. a kind of method of Fusarium oxysporum Cuba specialized form bacterial strain rejuvenation, which is characterized in that include the following steps：
1) by pathogenicity decline bacterial strain tieback to sterile water planting rooting tube plantlet.Aseptically, according to 1:10~15 ratio
The spore suspension of pathogenicity decline bacterial strain is added the fluid nutrient medium of sterile water planting rooting tube plantlet and shaken up by example；
2) after tieback 3~4 weeks, the separation for having the rooting tube plantlet of disease symptom to carry out pathogen is chosen.Aseptically from
The rooting tube plantlet base portion of above-mentioned tool classical symptom cuts bulb thin slice, is directly inoculated into culture medium culture；
3) after bacterium colony is grown, several bacteria cakes aseptically is cut along colony edge, are put into containing 0.05%~0.10%
In the sterile water of Tween-20, shake up at a slow speed to get the spore suspension of 1 generation of rejuvenation bacterial strain is arrived, and detect spore number；
4) aseptically, according to the method for step 1), by rejuvenation 1 generation bacterial strain tieback to sterile water planting rooting tube plantlet；
5) after tieback 3~4 weeks, the spore suspension of 2 generation of rejuvenation bacterial strain is arrived according to step 2)~step 3) method；
6) aseptically, according to the method for step 1), by rejuvenation 2 generation bacterial strain tieback to sterile water planting rooting tube plantlet；
7) 3-4 weeks after the 3rd tieback, the spore suspension of 3 generation of rejuvenation bacterial strain is arrived according to step 2)~step 3) method
Liquid, and the pathogenicity of rejuvenation 3 generation bacterial strain is measured.
2. the method according to claim 1, wherein spore in fluid nutrient medium after inoculation described in step 1)
Final concentration of the 10 of son5A spore/mL.
3. the method according to claim 1, wherein the rooting tube plantlet of disease symptom described in step 1) is
Yellowing leaf or withered rooting tube plantlet.
4. the method according to claim 1, wherein bulb thin slice described in step 2) is cut from yellowing leaf
Or withered rooting tube plantlet base portion.
5. the method according to claim 1, wherein culture medium described in step 2) is PDA culture medium.
6. the method according to claim 1, wherein laterally to cut bulb thin for rooting tube plantlet base portion in step 2)
Piece.
7. the method according to claim 1, wherein bacteria cake diameter described in step 3) is 5~7mm.
8. described in any item methods according to claim 1~7, which is characterized in that the bacterial strain is Fusarium oxysporum Cuba
Specialized form bacterial strain.
9. application of the method according to any one of claims 1 to 8 in bacterial strain rejuvenation.
10. application according to claim 9, the bacterial strain is Fusarium oxysporum Cuba specialized form bacterial strain.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112608852A (en) * | 2021-01-06 | 2021-04-06 | 袁隆平农业高科技股份有限公司 | Inoculation and propagation method of fusarium verticillioides |
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| CN104120084A (en) * | 2014-07-15 | 2014-10-29 | 云南农业大学 | Metarhizium flavoviride MFYY090714 and applications thereof |
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2018
- 2018-06-15 CN CN201810623456.1A patent/CN108865896A/en active Pending
Patent Citations (3)
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| CN101451108A (en) * | 2008-12-31 | 2009-06-10 | 云南农业大学 | Verticillium lecanii for preventing and controlling fly type pests and use thereof |
| CN103756913A (en) * | 2013-12-24 | 2014-04-30 | 华南农业大学 | Isaria fumosorosea |
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Non-Patent Citations (3)
| Title |
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| C. HOUBIN,ET AL.: "Screening of banana clones for resistance to fusarium wilt in China", 《ADVANCING BANANA AND PLANTAIN R&D IN ASIA AND THE PACIFIC》 * |
| 尹萃耘: "《农用微生物》", 31 December 1979 * |
| 广东农林学院微生物教研组: "《农业微生物学》", 30 June 1975 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112608852A (en) * | 2021-01-06 | 2021-04-06 | 袁隆平农业高科技股份有限公司 | Inoculation and propagation method of fusarium verticillioides |
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