CN105925488A - Preparation method of strawberry verticillium dahlia spore suspension - Google Patents
Preparation method of strawberry verticillium dahlia spore suspension Download PDFInfo
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- CN105925488A CN105925488A CN201610321648.8A CN201610321648A CN105925488A CN 105925488 A CN105925488 A CN 105925488A CN 201610321648 A CN201610321648 A CN 201610321648A CN 105925488 A CN105925488 A CN 105925488A
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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
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Abstract
The invention discloses a preparation method of a strawberry verticillium dahlia spore suspension. The method comprises the following steps of collecting strawberry verticillium dahlia infected plants; performing separation and purification; screening out pathogenic bacteria of the strawberry verticillium wilt; inoculating the separated verticillium dahlia into a PDA culture medium to perform plate culture and activation; taking down an agar block containing activated pathogenic bacteria; adding the agar block into a mixture formed by a liquid VL culture medium and 2.0-percent saccharose for shake culture; performing gauze filtering and filter paper vacuum pumping filtering for removing rest hyphae to obtain filter liquid for use; centrifuging filter liquid; abandoning supernate; and performing resuspension and sedimentation by 2ml of sterile water to obtain the strawberry verticillium dahlia spore suspension. The liquid culture medium inoculation and culture mode is used, so that the full growth all over the whole liquid culture medium can be fast achieved; and the labor consumption and the material consumption are reduced. The preparation method has the advantages that the inoculation amount is small; the spore generation speed is high; the growth is fast; the differences of spore quantities prepared from the same pathogenic bacteria in the same batch and different batches are small; and the method stability is high.
Description
Technical field
The invention belongs to microbial technology field, the preparation of a kind of strawberry Verticillium wilt bacterium spore suspension
Method.
Background technology
Strawberry Verticillium wilt is the intractable soil-borne disease that strawberry cultivating district of China generally occurs, the most susceptible very difficult
It is controlled with medicament.Shown by continuous qualification result: in the Changfeng County, Anhui, township of the famous Fructus Fragariae Ananssae of China
And planting base Jurong City that Jiangsu Province is important, the pathogen of strawberry Verticillium wilt is mainly Fusarium oxysporum.
At present, the Resistance Identification of the verticillium wilt of strawberry cultivars mainly uses disease nursery identification method, but due to disease
Between difference plot, garden, pathogenic bacteria concentration difference is bigger and the impact of frequent climate causes different year same kind grass
There is bigger difference in the resistance of certain kind of berries verticillium wilt, thus affects the accuracy of Disease Resistance Identification.
At present, artificial infection idenfication strawberry Verticillium wilt disease resistance mainly uses solid PDA medium to cultivate
This pathogen.After its natural birth spore, inject 3～5ml by having to length in the PDA culture medium of spore
Sterilized water；Fully after concussion, draw containing spore and the suspension of mycelia, by obtaining spore after filter paper filtering
Fullness over the chest during pregnancy supernatant liquid.But in actual operating process, owing to the cause of disease Fusarium oxysporum of strawberry Verticillium wilt exists
Grow very fast and actual sporulation quantity on solid PDA medium relatively fewer, and major part is mycelia, from
And cause needing to prepare a large amount of solid PDA medium to obtain sufficient concentrations of spore suspension.Additionally, its
Its some liquid such as looks into Bick and YPA culture medium etc., and the most generally to there is sporulation quantity less, mycelial growth mistake
The feature such as prosperous.Especially when needing to inoculate the bigger Fructus Fragariae Ananssae colony of quantity, need to consume substantial amounts of manpower and
Material resources prepare solid plate culture medium, on the other hand the most easily add the contamination probability of solid medium.
Therefore, the most more efficiently preparation is a large amount of and has the strawberry Verticillium wilt bacterium spore suspension of higher concentration and is
The technical problem that one letter is to be solved.
Summary of the invention
The present invention is in order to overcome the deficiency existing for above-mentioned prior art, it is provided that a kind of strawberry Verticillium wilt bacterium spore
The preparation method of suspension.
The purpose of the present invention can be achieved through the following technical solutions:
The preparation method of a kind of strawberry Verticillium wilt bacterium spore suspension, said method comprising the steps of:
(1) strawberrying verticillium wilt diseased plant, is primarily characterized in that blade exists big lobule and leaf curling
Phenomenon, is good for intersection in sick sample basal part of stem disease and cuts 10mm2Piece of tissue, 75% alcohol disinfecting 30s, aseptic
Water rinses 3 times, and aseptic absorbent paper is blotted, and is seeded in PDA culture medium, cultivates 3～4 under the conditions of 25 DEG C
Switching purification after it, 4 DEG C of Freezing Glycerines save backup；
(2) the verticillium wilt pathogenic bacteria of separation is inoculated on PDA culture medium flat plate, in 24 DEG C under dark condition
Constant temperature culture 6-7d；
(3) by the verticillium wilt pathogen of activation in step (2), add in the sucrose of fluid medium+2.0%, in
Under the conditions of 26 DEG C, isothermal vibration (130rpm/min) cultivates 6-7d, by culture fluid through filtration, vacuumizing filtration
Removing remaining mycelia, filtrate is standby；
(4) by step (3) filtrate centrifugal 30min under the conditions of 10000rmp/min, remove supernatant, use
The resuspended precipitation of 2ml sterilized water, i.e. obtains verticillium wilt pathogen spore suspension.
The formula of described step (2) PDA culture medium is: Rhizoma Solani tuber osi 200g, glucose 20g, agar 15-20g,
Pure water 1000ml, operating procedure: by peeling potatoes, cut fritter, put in beaker, add water 800ml,
Boil 30min, filter and remove Rhizoma Solani tuber osi residue, in filtrate, add glucose, agar, melt, be settled to
1000ml, 121 DEG C of high pressure steam sterilization 20min of last solution, cool down standby.
The formula of described step (3) fluid medium is: citrate dihydrate trisodium 2.6g, potassium nitrate 2.52g,
Ammonium dihydrogen phosphate 2.88g, potassium dihydrogen phosphate 1.6g, Magnesium sulfate heptahydrate 0.2g, calcium chloride dihydrate solution 0.1g,
Trace element solution 0.1ml, concentration are biotin solution 0.05ml of 0.1mg/ml, finally add pure water constant volume
To 1000ml.
Wherein, trace element solution formula is: water 95ml, citric acid monohydrate 5g, zinc sulphate heptahydrate 5g, six
Water ferric sulfate hydrate ammonium 1g, copper sulphate pentahydrate 250mg, manganese sulfate monohydrate 50mg, boric acid 50mg, two water
Sodium molybdate 50mg.
Described step (3) is filtered into three pull-up fat filtered through gauze.
Beneficial effects of the present invention: the present invention uses fluid medium inoculation and training method, it is possible to the longest
Full whole fluid medium, more than conventional center plating mode produces spore amount, only need a small amount of liquid training
Support base, save the substantial amounts of solid medium for cultivating pathogen, thus save man power and material
Consumption.Inoculum concentration of the present invention is few, and the speed that spore produces is fast, growth is fast, the same batch of same pathogenic bacteria, no
With preparing between batch, spore amount difference is little, and method stability is more preferable.
Detailed description of the invention
Embodiment 1
The preparation method of a kind of strawberry Verticillium wilt bacterium spore suspension, comprises the following steps:
1, the preparation of PDA culture medium:
Formula: peeled potatoes: 200g
Glucose: 20g
Agar: 15-20g
Pure water: 1000ml
Operating procedure: by peeling potatoes, cuts fritter, puts in beaker, and add water 800ml, boils 30min,
Filter and remove Rhizoma Solani tuber osi residue, in filtrate, add glucose, agar, melt, be settled to 1000ml,
121 DEG C of high pressure steam sterilization 20min of rear solution, cool down standby.
2, the formula of liquid VL culture medium is:
Citrate dihydrate trisodium 2.6g
Potassium nitrate 2.52g
Ammonium dihydrogen phosphate 2.88g
Potassium dihydrogen phosphate 1.6g
Magnesium sulfate heptahydrate 0.2g
Calcium chloride dihydrate solution 0.1g
Trace element solution 0.1ml
Concentration is biotin solution 0.05ml of 0.1mg/ml, finally adds pure water and is settled to 1000ml.
Wherein, trace element solution formula is: water 95ml, citric acid monohydrate 5g, zinc sulphate heptahydrate 5g, six
Water ferric sulfate hydrate ammonium 1g, copper sulphate pentahydrate 250mg, manganese sulfate monohydrate 50mg, boric acid 50mg, two water
Sodium molybdate 50mg.
3, strawberrying verticillium wilt diseased plant, is good for intersection in sick sample basal part of stem disease and cuts 10mm2Piece of tissue,
75% alcohol disinfecting 30s, rinsed with sterile water 3 times, aseptic absorbent paper is blotted, and is seeded in PDA culture medium,
Switching purification after cultivating 3～4 days under the conditions of 25 DEG C, 4 DEG C of Freezing Glycerines save backup；
4, the verticillium wilt pathogenic bacteria of separation is inoculated on PDA culture medium flat plate, in 24 DEG C of perseverances under dark condition
Temperature cultivates 6-7d；
5, by the verticillium wilt pathogen of above-mentioned activation, add in the sucrose of fluid medium+2.0%, in 26 DEG C of conditions
Lower isothermal vibration cultivates 6-7d, removes remaining by culture fluid through three pull-up fat filtered through gauze, vacuumizing filtration
Mycelia, filtrate is standby；
6, by above-mentioned filtrate centrifugal 30min under the conditions of 10000rmp/min, remove supernatant, aseptic with 2ml
The resuspended precipitation of water, i.e. obtains verticillium wilt pathogen spore suspension.
Above content is only citing made for the present invention and explanation, affiliated those skilled in the art
Described specific embodiment is made various amendment or supplements or use similar mode to substitute, as long as
Without departing from inventing or surmounting scope defined in the claims, protection scope of the present invention all should be belonged to.
Claims (4)
1. the preparation method of a strawberry Verticillium wilt bacterium spore suspension, it is characterised in that described method includes
Following steps:
(1) strawberrying verticillium wilt diseased plant, is good for intersection in sick sample basal part of stem disease and cuts 10mm2Piece of tissue,
75% alcohol disinfecting 30s, rinsed with sterile water 3 times, aseptic absorbent paper is blotted, and is seeded in PDA culture medium,
Switching and purification after cultivating 3～4 days under the conditions of 25 DEG C, 4 DEG C of Freezing Glycerines save backup；
(2) the verticillium wilt pathogenic bacteria of separation is inoculated on PDA culture medium flat plate activates, dark condition
Under in 24 DEG C of constant temperature culture 6-7d；
(3) step (2) will be placed in the sugarcane of liquid VL culture medium+2.0% containing the agar block activating pathogenic bacteria
In sugar, under the conditions of 26 DEG C, isothermal vibration cultivates 6-7d, is gone through filtered through gauze, vacuumizing filtration by culture fluid
Except remaining mycelia, filtrate is standby；
(4) by step (3) filtrate centrifugal 30min under the conditions of 10000rmp/min, remove supernatant, use
The resuspended precipitation of 2ml sterilized water, i.e. obtains verticillium wilt pathogen spore suspension.
The preparation method of strawberry Verticillium wilt bacterium spore suspension the most according to claim 1, its feature exists
In, the formula of described step (2) PDA culture medium is: Rhizoma Solani tuber osi 200g, glucose 20g, agar 15-20g,
Pure water 1000ml, operating procedure is: by peeling potatoes, cut fritter, puts in beaker, and add water 800ml,
Boil 30min, filter and remove Rhizoma Solani tuber osi residue, in filtrate, add glucose, agar, melt, be settled to
1000ml, 121 DEG C of high pressure steam sterilization 20min of last solution, cool down standby.
The preparation method of strawberry Verticillium wilt bacterium spore suspension the most according to claim 1, its feature exists
In, the formula of described step (3) liquid VL culture medium is: citrate dihydrate trisodium 2.6g, potassium nitrate 2.52g,
Ammonium dihydrogen phosphate 2.88g, potassium dihydrogen phosphate 1.6g, Magnesium sulfate heptahydrate 0.2g, calcium chloride dihydrate solution 0.1g,
Trace element solution 0.1ml, concentration are biotin solution 0.05ml of 0.1mg/ml, finally add pure water constant volume
To 1000ml；
Wherein, trace element solution formula is: water 95ml, citric acid monohydrate 5g, zinc sulphate heptahydrate 5g, six
Water ferric sulfate hydrate ammonium 1g, copper sulphate pentahydrate 250mg, manganese sulfate monohydrate 50mg, boric acid 50mg, two water
Sodium molybdate 50mg.
The preparation method of strawberry Verticillium wilt bacterium spore suspension the most according to claim 1, its feature exists
In, described step (3) is filtered into three pull-up fat filtered through gauze, then uses evacuation filter paper filtering to remove
Mycelia in culture medium.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108034593A (en) * | 2018-01-31 | 2018-05-15 | 北京农学院 | A kind of fungi culture medium and its application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102443544A (en) * | 2010-10-14 | 2012-05-09 | 河南省农业科学院 | Separation and purification method of sesame wilt germs |
| CN102732596A (en) * | 2011-04-01 | 2012-10-17 | 华中农业大学 | Cotton verticillium wilt resistant seedling stage identification method |
-
2016
- 2016-05-16 CN CN201610321648.8A patent/CN105925488A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102443544A (en) * | 2010-10-14 | 2012-05-09 | 河南省农业科学院 | Separation and purification method of sesame wilt germs |
| CN102732596A (en) * | 2011-04-01 | 2012-10-17 | 华中农业大学 | Cotton verticillium wilt resistant seedling stage identification method |
Non-Patent Citations (1)
| Title |
|---|
| 于红梅等: "草莓枯萎病菌的分离、鉴定及生物学特性", 《江苏农业科学》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108034593A (en) * | 2018-01-31 | 2018-05-15 | 北京农学院 | A kind of fungi culture medium and its application |
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Application publication date: 20160907 |
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