CN114836327A - Method for rapidly promoting growth of Cryptosphaeria pullmanensis H2 - Google Patents
Method for rapidly promoting growth of Cryptosphaeria pullmanensis H2 Download PDFInfo
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- 230000001737 promoting effect Effects 0.000 title claims abstract description 21
- 238000000034 method Methods 0.000 title claims abstract description 19
- 241001231297 Cryptosphaeria pullmanensis Species 0.000 title claims description 25
- 239000000706 filtrate Substances 0.000 claims abstract description 38
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 34
- 229920001817 Agar Polymers 0.000 claims abstract description 30
- 239000008272 agar Substances 0.000 claims abstract description 30
- 238000001914 filtration Methods 0.000 claims abstract description 7
- 235000009496 Juglans regia Nutrition 0.000 claims abstract description 6
- 235000020234 walnut Nutrition 0.000 claims abstract description 6
- 244000052769 pathogen Species 0.000 claims abstract description 5
- 230000001717 pathogenic effect Effects 0.000 claims abstract description 5
- 238000012258 culturing Methods 0.000 claims abstract description 4
- 240000007049 Juglans regia Species 0.000 claims abstract 2
- 241001231282 Cryptosphaeria Species 0.000 claims description 20
- 239000001963 growth medium Substances 0.000 claims description 6
- 239000008223 sterile water Substances 0.000 claims description 5
- 238000004090 dissolution Methods 0.000 claims description 2
- 238000011160 research Methods 0.000 abstract description 7
- 239000000463 material Substances 0.000 abstract description 4
- 241000233866 Fungi Species 0.000 abstract description 2
- 238000012136 culture method Methods 0.000 abstract description 2
- 230000000052 comparative effect Effects 0.000 description 22
- 239000002609 medium Substances 0.000 description 7
- 230000001580 bacterial effect Effects 0.000 description 5
- 244000052616 bacterial pathogen Species 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- 241000758789 Juglans Species 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 238000007789 sealing Methods 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 241001337994 Cryptococcus <scale insect> Species 0.000 description 2
- 238000010009 beating Methods 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000002503 metabolic effect Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 241000219000 Populus Species 0.000 description 1
- 241000124033 Salix Species 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000000498 cooling water Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000003973 irrigation Methods 0.000 description 1
- 230000002262 irrigation Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
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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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- 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
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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Abstract
The invention discloses a method for rapidly promoting the growth of cryptosphaerianulnensis H2, and particularly relates to the field of fungus culture. The method comprises the steps of collecting conidia angle of the walnut rot pathogen, dissolving the conidia angle, filtering and separating to obtain a filtrate of the conidia angle, and culturing the cryptosphaerianulmanensis H2 strain in an incubator by using water agar containing the filtrate of the conidia angle. The culture method can accelerate the growth speed of Cryptosphaerianulenensis H2, and hyphae grow more densely by adding the conidium angle H2, so that good research materials can be provided for subsequent related researches, and time and labor are saved.
Description
Technical Field
The invention relates to the field of fungus culture, in particular to a method for rapidly promoting the growth of Cryptosphaeria pullmannsis H2.
Background
Northwest juncture in China, around the edges and hills, far from the ocean, are provinces with the largest occupied area in China, and according to records of Xinjiang arbors, shrubs and half shrubs, 55 varieties of 183 genera of 871 in the 75 families in Xinjiang arbor, shrub and half shrub books are provided. The arbor, shrub and some rare or endangered plants are the material basis for selecting and breeding excellent trees in Xinjiang and are valuable tree species resources in Xinjiang. However, due to the special geographical location of Xinjiang, many plants are damaged by various germs because of the restriction of natural conditions such as geography, landform, climate, soil and irrigation. Various pathogenic bacteria cause withered numbers and even death of the whole plant, and the development of agriculture, forestry, ecological environment and other aspects in China is severely restricted. The research on pathogenic bacteria and the finding of proper prevention and treatment measures are urgent.
Cryptosphaeria (Cryptosphaeria pullmanensis) is a main pathogenic bacterium causing the poplar and willow canker reported at home and abroad. However, the growth of cryptococcus is slow, and pure culture is not easy to obtain. Finding out the method capable of promoting the rapid growth of the strain lays a certain foundation for the smooth development of the research related to the strain, thereby having important significance for protecting the current ecological environment.
Disclosure of Invention
Therefore, the invention provides a method for rapidly promoting the growth of Cryptosphaeria pullmannsis H2, so as to solve the problem that the subsequent research is influenced by the slow growth of Cryptosphaeria pullmannsis H2.
In order to achieve the above purpose, the invention provides the following technical scheme:
the method for rapidly promoting the growth of Cryptosphaeria pullmannsis H2 comprises the steps of collecting conidia of the walnut rot pathogen, dissolving the conidia, filtering and separating to obtain a conidia filtrate, and culturing the Cryptosphaeria pullmannsis H2 strain in an incubator by using water agar containing the conidia filtrate.
Further, the filtration is performed by using a 0.22 μm filter.
Further, the dissolution was 0.05g of conidia horn dissolved in 500. mu.L of sterile water.
Further, the preparation method of the water agar containing the conidium horn filtrate comprises the steps of shaking the filtrate into the water agar, inverting the water agar containing the filtrate, and solidifying the culture medium to obtain the water agar containing the conidium horn filtrate.
Further, in the water agar containing the conidia angle filtrate, the ratio of the filtrate to the water agar is that every 200. mu.L of the filtrate is put into 150mL of the water agar.
Further, the water agar plates containing the filtrate were 10 plates per 150mL of water agar.
Furthermore, the culture condition of the incubator is that the temperature is 26-28 ℃, and the culture is performed alternately in light and shade.
Further, the culture condition of the incubator is 26-28 ℃, and dark culture is carried out.
The invention has the following advantages:
the culture method can accelerate the growth speed of Cryptosphaeria pullmanensis H2 and make the hypha grow more densely. The growth of the strain is slower than that of the common strain, if the conidium angle which is a free material which is very easy to obtain is added, the growth of the strain can be accelerated, good research materials can be provided for subsequent related researches, the cost is low, and the time and the labor are saved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.
The structures, ratios, sizes, and the like shown in the present specification are only used for matching with the contents disclosed in the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions that the present invention can be implemented, so that the present invention has no technical significance, and any structural modifications, changes in the ratio relationship, or adjustments of the sizes, without affecting the effects and the achievable by the present invention, should still fall within the range that the technical contents disclosed in the present invention can cover.
FIG. 1 is a colony diagram of Cryptosphaeria pullmanensis H2 strain cultured on day 4 in example 1 and comparative example 1;
FIG. 2 is a 7-day colony diagram of Cryptosphaeria pullmanensis H2 cultured in example 1 and comparative example 1;
FIG. 3 is a 10-day colony diagram of Cryptosphaeria pullmanensis H2 cultured in example 1 and comparative example 1;
FIG. 4 is a 15-day colony diagram of Cryptosphaeria pullmanensis H2 cultured in example 1 and comparative example 1;
FIG. 5 is a graph showing the measurement of colony growth diameter after adding conidia horns to the water agar medium of example 1 and not adding conidia horns to comparative example 1, which are provided by the present invention;
FIG. 6 is a colony map of Cryptosphaeria pullmanensis H2 cultured in example 2 and comparative example 2 on different days; wherein, 1 represents day 3; 2 represents day 5; 3 represents day 7; 4 represents day 10;
FIG. 7 is a graph showing the measurement of colony growth diameter after adding conidia horns to water agar medium in example 2 and not adding conidia horns in comparative example 2, which are provided by the present invention;
in the figure, ABC is a diagram of the filtrate of comparative example 1 without addition of conidia horns; DEF is a picture of conidia horn filtrate added in example 1; g is the colony pattern of the culture with the addition of the conidial of example 2, and H is the colony pattern of the culture without the addition of the conidial filtrate of comparative example 2.
Detailed Description
The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1A method for rapidly promoting the growth of Cryptosphaeria pullmanensis H2
The method comprises the following steps:
step one, sampling
Collecting conidium angles of the walnut rot germs; cleaning with sterilized water, centrifuging at 12000r/min for 3min, removing supernatant, cleaning repeatedly for 3 times, and removing supernatant;
step two, separation and culture
Taking the lower layer conidium horn into a new centrifuge tube, taking sterile water on a sterile operation table, dissolving the conidium horn in the centrifuge tube (0.05g of conidium is dissolved in 500 mu L of sterile water), sucking the conidium horn dissolving liquid by using a syringe, filtering by using a 0.22 mu m filter, putting the filtrate into the sterile centrifuge tube, putting every 200 mu L of the filtrate into 150mL of water agar, shaking uniformly, and pouring every 150mL of water agar into 10 plates. When the culture medium is solidified, beating a Cryptosphaeria pullmanensis H2 bacterial cake, and placing the bacterial cake in the middle of the culture medium; placing in an incubator at the temperature of 26 ℃, and performing dark culture; and colony data was recorded for different days of culture.
Step three, ITS identification
After the culture is finished, directly sealing the spore suspension by a sealing film, sending the spore suspension to Shanghai workers at normal temperature for identification, wherein the ITS identification result is cryptococcus cryptosphaera Pullmanensis.
Step four, sequencing
The pure culture obtained from the second step was purified again to a single colony and sent to the Producer sequencing company, and the result was consistent, and was Cryptosphaeria monocytogenes.
Example 2
The embodiment of the invention provides a method for rapidly promoting the growth of Cryptosphaeria pullmannsis H2, which comprises the following steps:
the same operation as in example 1 was carried out except for the culture conditions: culturing at 26 deg.C under alternating light and dark for 12 hr; after the culture is finished, ITS identification and sequencing are carried out; the result was consistent with that of Cryptosphaeria pullmanensis.
Example 3
Firstly, 500 mu L of sterile water is taken from an aseptic operation table and put into a centrifuge tube to dissolve conidium horns, then a syringe is used for sucking conidium horn dissolving liquid (the liquid is yellow), then the liquid is filtered by a 0.22 mu m filter, filtrate is put into the aseptic centrifuge tube, a new 0.22 mu m filter is used for repeatedly filtering until the filtrate is colorless, colorless and transparent filtrate is put into the aseptic centrifuge tube, and finally every 200 mu L of filtrate is put into 150mL of cooling water agar to be uniformly shaken, and every 150mL of water agar is poured into 10 plates.
When the culture medium is solidified, beating a Cryptosphaeria pullmansensis H2 bacterial cake, and placing the bacterial cake in the middle of the culture medium; placing the mixture in an incubator at the temperature of 28 ℃ for dark culture.
Step two, ITS identification
Directly sealing the spore suspension with a sealing film, sending to Shanghai workers at normal temperature for identification, wherein the ITS identification result is Cryptosphaeria pullmanensis.
Step three, sequencing
The pure culture obtained from step one was purified again to a single colony and sent to the Producer sequencing company with consistent results, also Cryptosphaeria pullmanensis.
Comparative example 1
Comparative example is an aqueous agar medium without addition of a conidial filtrate, which was prepared in the same manner as in example 1 except that no conidial filtrate was added, and was placed in an incubator at a temperature of 26 ℃ and cultured in the dark.
Comparative example 2
The comparative example is a water agar medium without adding a spore horn filtrate, which is exactly the same as that of example 2 except that no spore filtrate was added, and the medium was incubated in an incubator at 26 ℃ and alternately in light and dark for 12 hours.
Test example 1
The colony size of the cultured Cryptosphaeria pullmanensis H2 strain of example 1 was measured on day 4, day 7, day 10 and day 15, respectively, and the data was recorded.
The colony sizes of the Cryptosphaeria pullmanensis H2 strain cultured in comparative example 1 were measured on days 4, 7, 10 and 15, respectively, and the data were recorded.
FIG. 1 is a DEF of example 1 showing a colony of Cryptosphaeria pullmanensis H2 strain on day 4 after addition of conidium filtrate; FIG. 2 is a DEF of example 1 showing a 7-day colony of Cryptosphaeria pullmanensis H2 strain cultured with the addition of a conidium filtrate; FIG. 3 is a DEF of example 1 showing a 10-day colony of Cryptosphaeria pullmanensis H2 strain cultured with the addition of a conidium filtrate; FIG. 4 is a DEF of example 1, showing a 15 th day colony of Cryptosphaeria pullmanensis H2 strain cultured with the addition of conidium filtrate;
FIG. 1 is a photograph showing the colony of Cryptosphaeria pullmanensis H2 cultured in comparative example 1 on day 4; FIG. 2 is an ABC map of a 7-day colony of Cryptosphaeria pullmanensis H2 cultured in comparative example 1; FIG. 3 is an ABC map of a 10-day colony of Cryptosphaeria pullmansensis H2 cultured in comparative example 1; FIG. 4 is an ABC map of a 15-day colony of Cryptosphaeria pullmansensis H2 cultured in comparative example 1.
FIG. 5 is a graph showing the measurement of colony growth diameter after adding conidia horns to water agar medium in example 1 and not adding conidia horns in comparative example 1.
Therefore, the metabolic components in the conidium angle of the walnut rot pathogen have the growth promoting effect, and the growth promoting effect is remarkable in 15 days.
Test example 2
The colony size of the cultured Cryptosphaeria pullmanensis H2 strain of example 2 was measured on days 3, 5, 7, 10 and 13, respectively, and the data was recorded.
The colony size of the cultured bacterium Cryptosphaeria pullmanensis H2 of comparative example 2 was measured on days 3, 5, 7, 10 and 13, respectively, and the data was recorded.
FIG. 6, G is a graph of colonies on day 10 in example 2, in which each line segment indicates the growth length of colonies on different days, and 1 indicates day 3; 2 represents day 5; 3 represents day 7; 4 represents day 10;
FIG. 6, H is a plot of colonies on day 10 of comparative example 2, in which each segment represents the growth length of colonies on different days, and 1 represents day 3; 2 represents day 5; 3 represents day 7; and 4 represents day 10.
FIG. 7 is a graph showing the measurement of colony growth diameter after adding conidia horns to the water agar medium in example 2 and not adding conidia horns in comparative example 2, which are provided by the present invention.
By measuring the diameter of the Cryptosphaeria pullmannsis H2 bacterial colony, it can be seen that the metabolic component in the conidia angle of the walnut rot pathogen has the growth promoting effect indeed, and the growth promoting effect is obvious in 15 days.
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (8)
1. The method for rapidly promoting the growth of Cryptosphaeria pullmannsis H2 is characterized by comprising the steps of collecting conidia angles of the walnut rot pathogen, dissolving the conidia angles, filtering and separating to obtain a filtrate of the conidia angles, and culturing the Cryptosphaeria pullmannsis H2 strain in an incubator by using water agar containing the filtrate of the conidia angles.
2. The method for rapidly promoting the growth of Cryptosphaeria pullmannsis H2 according to claim 1, wherein the filtration is performed by using a 0.22 μm filter.
3. The method for rapidly promoting the growth of Cryptosphaeria pullmannsis H2 according to claim 1, wherein the dissolution is 0.05g of conidia horn dissolved in 500 μ L of sterile water.
4. The method for rapidly promoting the growth of Cryptosphaeria pullmanensis H2 according to claim 1, wherein the water agar containing the conidia angle filtrate is prepared by shaking the filtrate into the water agar, pouring the water agar containing the filtrate, and allowing the culture medium to solidify.
5. The method for rapidly promoting the growth of Cryptosphaeria pullmannsis H2 according to claim 4, wherein the ratio of the filtrate to the water agar in the water agar containing the conidia horn filtrate is that every 200 μ L of the filtrate is placed in 150mL of the water agar.
6. The method for rapidly promoting the growth of Cryptosphaeria pullmannsis H2 according to claim 4, wherein the water agar plate containing the filtrate is 10 plates per 150mL of water agar.
7. The method for rapidly promoting the growth of Cryptosphaeria pullmannsis H2 according to claim 1, wherein the cultivation condition of the incubator is a temperature of 26-28 ℃ and alternate light and dark cultivation.
8. The method for rapidly promoting the growth of Cryptosphaeria pullmannsis H2 according to claim 1, wherein the cultivation condition of the incubator is a temperature of 26-28 ℃, and the incubator is a dark cultivation.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2550364A1 (en) * | 2010-03-24 | 2013-01-30 | Livefuels, Inc | Systems and methods for producing eicosapentaenoic acid and docosahexaenoic acid from algae |
| CN103627800A (en) * | 2013-11-14 | 2014-03-12 | 浙江天科高新技术发展有限公司 | Rapid detection method of environmental microorganisms |
| CN106085945A (en) * | 2016-08-24 | 2016-11-09 | 文山苗乡三七科技有限公司 | A kind of Radix Notoginseng Northern leaf spot bacterium conidium abductive approach |
| WO2020245438A1 (en) * | 2019-06-06 | 2020-12-10 | Instituto Superior De Agronomia | Plant treatment |
| CN113151043A (en) * | 2021-02-04 | 2021-07-23 | 塔里木大学 | Rhizobium DG3-1 for resisting cotton verticillium wilt and application thereof |
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- 2022-04-29 CN CN202210468596.2A patent/CN114836327A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2550364A1 (en) * | 2010-03-24 | 2013-01-30 | Livefuels, Inc | Systems and methods for producing eicosapentaenoic acid and docosahexaenoic acid from algae |
| CN103627800A (en) * | 2013-11-14 | 2014-03-12 | 浙江天科高新技术发展有限公司 | Rapid detection method of environmental microorganisms |
| CN106085945A (en) * | 2016-08-24 | 2016-11-09 | 文山苗乡三七科技有限公司 | A kind of Radix Notoginseng Northern leaf spot bacterium conidium abductive approach |
| WO2020245438A1 (en) * | 2019-06-06 | 2020-12-10 | Instituto Superior De Agronomia | Plant treatment |
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Application publication date: 20220802 |