CN104328059A - Mulberry surface corrosion-causing antagonistic bacteria screening method - Google Patents
Mulberry surface corrosion-causing antagonistic bacteria screening method Download PDFInfo
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- CN104328059A CN104328059A CN201410543580.9A CN201410543580A CN104328059A CN 104328059 A CN104328059 A CN 104328059A CN 201410543580 A CN201410543580 A CN 201410543580A CN 104328059 A CN104328059 A CN 104328059A
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- antagonistic fungi
- screening method
- mulberry fruit
- rotten
- bacterium colony
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/16—Yeasts; Culture media therefor
Abstract
The invention discloses a mulberry surface corrosion-causing antagonistic bacteria screening method which is as follows: 1, taking clean and intact mulberry, chopping, then placing in sterile physiological saline, shaking mixed liquid for culturing at constant temperature to obtain antagonistic bacteria liquid; 2, diluting the antagonistic bacteria liquid with the sterile physiological saline to obtain diluted liquid, taking 1mL diluted liquid to applying to a PDA (potato dextrose agar) culture medium, and culturing at constant temperature to obtain bacterial colony group; and 3, picking the obtained bacterial colony group, inoculating onto the PDA culture medium for pure culture, and repeatedly separating for 3-5 times to obtain antagonistic bacteria single colony. The screening method has the advantages of simple operation, strong selectivity, and better separation effect.
Description
Technical field
The invention belongs to microbial technology field, be specifically related to the screening method that a kind of mulberry fruit surface causes rotten Antagonistic Fungi.
Background technology
Mulberry fruit belongs to berry fruit, and epidermis is very thin, fruit soft and succulency, breathes vigorous, and the ripening stage is short, and mature fruit is plucked or very easily sustained damage in transportation; In addition the mulberry fruit ripening stage is on the occasion of hot weather, is subject to cause pathogeny imcrobe infection, causes a large amount of rotting.At present, for the aspect of antiseptic preservation of fruits and vegetables, mainly adopt chemical bactericide, though play fresh-keeping effect, potential harm is brought to the edibleness of fruits and vegetables.Seek health, the biological way of keeping fresh of environmental protection more and more receive publicity, wherein utilize the antagonistic action of microorganism carry out biological control more researcher pay much attention to.Screening can suppress mulberry fruit surface to cause the main diseases original of rotten bacterium, significant to the biological way of keeping fresh setting up effective mulberry fruit and relevant fruits and vegetables.
Summary of the invention
The object of this invention is to provide the screening method that a kind of mulberry fruit surface causes rotten Antagonistic Fungi, the method is easy and simple to handle, selectivity is strong, separating effect is excellent.
Mulberry fruit surface causes the screening method of rotten Antagonistic Fungi, comprises the following steps:
Step 1, the preparation of Antagonistic Fungi liquid: get clean intact mulberry fruit, chopping is placed in stroke-physiological saline solution, and by mixed solution constant-temperature shaking culture, gained nutrient solution is Antagonistic Fungi liquid;
Step 2, antagonism bacterium colony group cultivates: the dilution of step 1 gained Antagonistic Fungi liquid stroke-physiological saline solution is obtained diluent, and get 1mL diluent and coat on PDA substratum, constant temperature culture, obtains bacterium colony group;
Step 3, single bacterium colony is cultivated: picking step 2 gained bacterium colony group, is seeded in PDA substratum and carries out pure culture, be repeatedly separated 3 ~ 5 times, can obtain Antagonistic Fungi list bacterium colony.
As the further improvement of foregoing invention, in step 1 mixed solution, the mass concentration of mulberry fruit is 20 ~ 25%.
As the further improvement of foregoing invention, in step 1 shaking culture be 27 ~ 28 DEG C, cultivate 25 ~ 30min under 2500rpm oscillating condition.
As the further improvement of foregoing invention, in step 2, the concentration of gained diluent is respectively 10
-6, 10
-7, 10
-8.
As the further improvement of foregoing invention, in step 2, constant temperature culture cultivates 72 ~ 78h under 27 ~ 28 DEG C of conditions.
Advantage of the present invention and beneficial effect:
1. by with cause rotten bacterium Dual culture, verify that this pathogenic bacteria has and suppress to cause the effect of rotten bacteria growing, with strong points;
2. utilize the yeast culture medium containing beef extract-peptone to cultivate this pathogenic bacteria, observe its morphological specificity, tentatively can judge the source of screened microorganism;
3. the pathogenic bacteria attribute that molecular biology method screens for qualification provides material evidence.
Accompanying drawing explanation
Figure 1A is Antagonistic Fungi and cause rotten bacterium (chain lattice satisfy bacterium) Dual culture and inoculate and enclose substratum front elevation;
Figure 1B is Antagonistic Fungi and cause rotten bacterium (chain lattice satisfy bacterium) Dual culture and inoculate and enclose substratum back view;
Fig. 2 is Antagonistic Fungi thallospore figure;
Fig. 3 is phylogenetic tree analysis chart.
Embodiment
Embodiment 1
Mulberry fruit surface causes the screening method of rotten Antagonistic Fungi, comprises the following steps:
Step 1, the preparation of Antagonistic Fungi liquid: get clean intact mulberry fruit, chopping is placed in stroke-physiological saline solution, and obtain mixed solution, in mixed solution, the mass concentration of mulberry fruit is 20%; By mixed solution 28 DEG C, cultivate 25 ~ 30min under 2500rpm oscillating condition, gained nutrient solution is Antagonistic Fungi liquid.
Step 2, antagonism bacterium colony group cultivates: step 1 gained Antagonistic Fungi liquid stroke-physiological saline solution diluted, the concentration of gained diluent is respectively 10
-6, 10
-7, 10
-8, get 1mL diluent respectively and coat on PDA substratum, constant temperature culture 72 ~ 78h under 27 ~ 28 DEG C of conditions, obtains bacterium colony group.
Step 3, single bacterium colony is cultivated: picking step 2 gained bacterium colony group, is seeded in PDA substratum and carries out pure culture, be repeatedly separated 3 ~ 5 times, can obtain Antagonistic Fungi list bacterium colony.
Described PDA substratum contains 20% potato filtrate (peeling potatoes, stripping and slicing are boiled, and boil thoroughly well cooked but not mushy, four layers of filtered through gauze), 2% agar, 2% glucose, under natural pH, and 121 DEG C of sterilizing 20 ~ 25min.
Experimental example 1 proof test
Aseptically, Example 1 gained Antagonistic Fungi list bacterium colony, in the middle of dull and stereotyped PDA substratum, flat board is divided into two by line, cause rotten bacterium-chain lattice to satisfy bacterium bacterium block on the mulberry fruit surface that mid-line both sides equidistant inoculation size is identical, cultivate 5 ~ 7 days in 27 ~ 28 DEG C of incubators, observe inoculation circle growing state.As shown in FIG. 1A and 1B, two side chain lattice bacterium bacterium circle of satisfying does not expand to mid-line, illustrates that embodiment 1 gained Antagonistic Fungi list bacterium colony has suppression chain lattice significantly and to satisfy the effect of bacteria growing.
The morphologic observation of experimental example 2 Antagonistic Fungi
By embodiment 1 gained Antagonistic Fungi list colony inoculation on beef-protein medium (microzyme culture medium), as shown in Figure 2, gained bacterium colony is creamy white, bacterium colony surface is dim and smooth coarse, colony edge is smoothly in wavy, mycelia has gemmation phenomenon, can judge this cause of disease Pseudomonas yeast.
Experimental example 3 Antagonistic Fungi phylogenetic feature Molecular Identification
Adopt yeast bacterium genomic dna Rapid extraction test kit to extract antagonistic strain STb gene, design primer, ITS1:5 '-TCCGTAGGTGAACCTGCGG-3 ' according to yeast ribosomal gene conserved sequence 5.8S-ITSrDNA; ITS4:5 '-TCCTCCGCTTATTGATATGC-3 '.Through pcr amplification, the connection of pcr amplification product and pMD18-T carrier, cell transformation, gene cloning and sequencing, with GenBank database comparison equimolecular biological procedures, verifies the phylogenetic feature of this Antagonistic Fungi.As shown in Figure 3, acquired results illustrates that this Antagonistic Fungi is that yeast thrown by lock.
Claims (5)
1. mulberry fruit surface causes the screening method of rotten Antagonistic Fungi, it is characterized in that: comprise the following steps:
Step 1, the preparation of Antagonistic Fungi liquid: get clean intact mulberry fruit, chopping is placed in stroke-physiological saline solution, and by mixed solution constant-temperature shaking culture, gained nutrient solution is Antagonistic Fungi liquid;
Step 2, antagonism bacterium colony group cultivates: the dilution of step 1 gained Antagonistic Fungi liquid stroke-physiological saline solution is obtained diluent, and get 1mL diluent and coat on PDA substratum, constant temperature culture, obtains bacterium colony group;
Step 3, single bacterium colony is cultivated: picking step 2 gained bacterium colony group, is seeded in PDA substratum and carries out pure culture, be repeatedly separated 3 ~ 5 times, can obtain Antagonistic Fungi list bacterium colony.
2. mulberry fruit surface according to claim 1 causes the screening method of rotten Antagonistic Fungi, it is characterized in that: in step 1 mixed solution, the mass concentration of mulberry fruit is 20 ~ 25%.
3. mulberry fruit according to claim 1 surface causes the screening method of rotten Antagonistic Fungi, it is characterized in that: in step 1 shaking culture be 27 ~ 28 DEG C, cultivate 25 ~ 30min under 2500rpm oscillating condition.
4. mulberry fruit surface according to claim 1 causes the screening method of rotten Antagonistic Fungi, it is characterized in that: in step 2, the concentration of gained diluent is respectively 10
-6, 10
-7, 10
-8.
5. mulberry fruit surface according to claim 1 causes the screening method of rotten Antagonistic Fungi, it is characterized in that: in step 2, constant temperature culture cultivates 72 ~ 78h under 27 ~ 28 DEG C of conditions.
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| CN201410543580.9A CN104328059A (en) | 2014-10-14 | 2014-10-14 | Mulberry surface corrosion-causing antagonistic bacteria screening method |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108504578A (en) * | 2018-05-31 | 2018-09-07 | 江苏农林职业技术学院 | A kind of screening technique of clematis leaf spot Antagonistic Fungi |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102533564A (en) * | 2011-11-21 | 2012-07-04 | 滨州职业学院 | Method for screening bio-control trichoderma in corn seedling stage root rot period |
| CN104031839A (en) * | 2014-05-26 | 2014-09-10 | 江苏科技大学 | Method for screening rotten fungi on surface of mulberry |
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2014
- 2014-10-14 CN CN201410543580.9A patent/CN104328059A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102533564A (en) * | 2011-11-21 | 2012-07-04 | 滨州职业学院 | Method for screening bio-control trichoderma in corn seedling stage root rot period |
| CN104031839A (en) * | 2014-05-26 | 2014-09-10 | 江苏科技大学 | Method for screening rotten fungi on surface of mulberry |
Non-Patent Citations (1)
| Title |
|---|
| 王晓红等: "毛白杨内生真菌的分离与接抗菌的筛选", 《河北林果研究》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108504578A (en) * | 2018-05-31 | 2018-09-07 | 江苏农林职业技术学院 | A kind of screening technique of clematis leaf spot Antagonistic Fungi |
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Application publication date: 20150204 |
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