CN114214199B - Preservation method of Isaria cicadae spore powder - Google Patents
Preservation method of Isaria cicadae spore powder Download PDFInfo
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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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Abstract
The application discloses a preservation method of Isaria cicadae spore powder, belonging to the technical field of microbial strain preservation. The method comprises the following steps: obtaining spore powder of the corynespora cicadae, and hermetically preserving the corynespora cicadae in the form of spore powder at the ultralow temperature of-80 ℃ or the low temperature of 4 ℃. According to the application, the cicada fungus is preserved in the form of spore powder, the spore powder is placed in different temperature environments, the preservation conditions such as detection time period and the like are combined with standards such as mycelium growth, spore germination, mycelium biomass in fungus suspension and infection capability of tussah pupa and the like to evaluate, and finally the spore powder is preserved at the ultralow temperature of-80 ℃ or the low temperature of 4 ℃, so that the activity of the spore powder can be kept for more than 24 months, the spore powder has the activity of infecting insects, and the spore powder is an optimal scheme for long-term preservation, and has important significance in filling the technical blank in the strain preservation field of the cicada fungus and providing high-quality and stable cicada fungus germplasm resources.
Description
Technical Field
The application relates to the technical field of microbial strain preservation, in particular to a preservation method of Isaria cicadae spore powder.
Background
Isaria cicadae (Isaria cicadae) belongs to the fungi of the genus Isaria of the order Ascomycota, hypocreaceae. From ancient times to date, most medical books widely acknowledged that the name is only the name of 'cordyceps sobolifera', and the characteristics of the cordyceps sinensis can be summarized most accurately and vividly, so that the cordyceps sinensis is a traditional and precious Chinese medicinal material in China, and the use history is long. Compared with the growth environment of Cordyceps, the Isaria cicadae has wider distribution, and mainly appears in the Yangtze river and the south of China, including Sichuan, jiangsu, zhejiang, fujian, anhui, guangdong, yunnan and other provinces. The wild and artificially cultured Isaria cicadae have rich nutrition, and can be used for separating and extracting many active ingredients beneficial to human health such as amino acid, adenosine, ergosterol, cordycepin, etc.
Research on the artificial culture technology, biocontrol effect and the like of the corynespora cicadae has been widely conducted. However, in production practice, a common problem encountered by cultivation technicians is that the artificially isolated strain is easy to generate production property variation on a culture medium, namely strain degeneration phenomenon is commonly known, the infection capability of the degenerated strain is reduced, and the corynespora cicadae has the advantages of high safety and large spore yield, so that the corynespora cicadae is a broad-spectrum insect-producing fungus with higher pathogenicity on pests, is very suitable for being used as a pesticide to prevent and treat harmful insects, but the spore preparation of the corynespora cicadae is greatly limited in production application due to the limiting factors of slower pest knockdown speed, dependence on humidity, intolerable spore storage and the like other biocontrol fungi. Therefore, it is important to effectively store spores and increase their infectivity by preservation means.
Disclosure of Invention
The application aims to provide a preservation method of spore powder of corynespora cicadae, which solves the problems in the prior art, and the spore can keep activity for more than 24 months under refrigeration condition of minus 80 ℃ and 4 ℃ by preserving the strain of the corynespora cicadae in the form of spore powder, has the capability of infecting insects, and is a high-quality preservation method suitable for preserving the strain of the corynespora cicadae for a long time.
In order to achieve the above object, the present application provides the following solutions:
the application provides a preservation method of Isaria cicadae spore powder, which comprises the following steps:
obtaining spore powder of the corynespora cicadae, and hermetically preserving the corynespora cicadae in the form of spore powder at the ultralow temperature of-80 ℃ or the low temperature of 4 ℃.
Preferably, the method for obtaining the spore powder of the corynespora cicadae comprises the following steps:
(1) Inoculating the Isaria cicadae on a solid modified potato glucose culture medium, and culturing until hyphae grow out; the solid modified potato dextrose culture medium comprises the following components in parts by weight: 200 parts of potato, 20 parts of glucose, 3 parts of peptone, 1 part of magnesium sulfate heptahydrate, 2 parts of potassium dihydrogen phosphate, 0.025 part of vitamin B,16 parts of agar and 1000 parts of water;
(2) Transferring the mycelium and the spores to a liquid modified potato dextrose medium, and culturing to obtain a bacterial suspension; the liquid modified potato glucose culture medium is the solid modified potato glucose culture medium, and agar is not added in the solid modified potato glucose culture medium;
(3) Inoculating the bacterial suspension to a wheat culture medium, and culturing to obtain spore powder of the corynespora cicadae; the wheat culture medium comprises the following components in parts by weight: 15 parts of wheat and 22.5 parts of liquid modified potato dextrose medium.
Preferably, in the step (1), after the culturing condition is that the culturing is carried out in darkness at 23 ℃ for 3 days, the culturing is continued under the illumination condition until the mycelium grows to contact the inner wall of the culture vessel.
Preferably, in the step (2), the culture condition is 23℃and 150rpm for 3 days to obtain a bacterial suspension.
Preferably, in the step (3), the culture condition is that the obtained bacterial suspension is inoculated in the wheat culture medium according to the inoculum size of 5 percent, and is cultured in the dark at the temperature of 23 ℃ for 3 days, then is subjected to illumination culture for 17 days, and then the spore powder of the corynespora cicadae is collected.
The application also provides application of the preservation method in preventing degradation of the corynespora cicadae.
The application discloses the following technical effects:
the spore powder is placed in different temperature environments, preservation conditions such as detection time period and the like are combined with standards such as hypha growth condition, spore germination condition, mycelium biomass in bacterial suspension and infection capability of tussah pupa to evaluate, so as to formulate an optimal preservation scheme, and draw the following conclusion: the corynespora cicadae strain is preserved in the form of spore powder, and can be preserved for a short period of time under the condition of room temperature, and the preservation period can reach 3 months; preserving at-20deg.C under refrigeration for 6 months, the spores still can keep activity; the spores can remain active for more than 24 months under refrigeration conditions of-80 ℃ and 4 ℃ and have the capability of infecting insects, and are the optimal temperature for long-term preservation. The application mainly aims at the technical blank in the field of the preservation of the corynespora cicadae strains, provides a high-quality preservation method of the corynespora cicadae strains, and provides a novel method for guaranteeing high-quality and stable corynespora cicadae germplasm resources.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the growth of spores stored at different temperatures during different periods of preservation;
FIG. 2 shows the growth of spores at different storage periods at a suitable temperature and suitable storage temperature;
FIG. 3 shows spore germination of spores stored at different temperatures and at different storage periods;
FIG. 4 shows spore germination of spores at different storage periods for spores of suitable storage temperature;
FIG. 5 shows mycelium formation of Isaria cicadae powder stored at different temperatures for 6 months;
FIG. 6 is the effect of Isaria cicadae spore powder on hyphal biomass stored at different temperatures for 6 months;
FIG. 7 shows a situation in which Isaria cicadae spore powder stored at different temperatures for 6 months infects tussah pupae;
FIG. 8 is the effect of Isaria cicadae spore powder stored at different temperatures for 6 months on tussah pupa infestation activity;
FIG. 9 shows infection of tussah pupae with Isaria cicadae spore powder stored at-80deg.C and 4deg.C for a long period of time;
FIG. 10 is a graph showing the effect of storing Isaria cicadae spore powder at-80℃and 4℃for a long period of time on the activity of tussah pupae infestation;
FIG. 11 shows the effect of 7 and 14 days of incubation at different temperatures on the water content of the medium;
FIG. 12 shows the effect of 3 days, 7 days and 14 days of storage at different temperatures on hypha growth.
Detailed Description
Various exemplary embodiments of the application will now be described in detail, which should not be considered as limiting the application, but rather as more detailed descriptions of certain aspects, features and embodiments of the application.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the application. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the application described herein without departing from the scope or spirit of the application. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present application. The specification and examples of the present application are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
Example 1
1. Experimental materials and methods
1.1 test materials
1.1.1 test strains
The test strain was isolated and purified from the Isaria cicadae entity of Fujian and deposited in the plant protection institute of university of Shenyang, mycological laboratory.
1.1.2 test Medium
Solid modified potato dextrose medium (modified PDA): 200 parts of potato, 20 parts of glucose, 3 parts of peptone, 1 part of magnesium sulfate heptahydrate, 2 parts of potassium dihydrogen phosphate, 0.025 part of vitamin B,16 parts of agar and 1000 parts of water.
Liquid modified potato dextrose medium (modified PD): 200 parts of potato, 20 parts of glucose, 3 parts of peptone, 1 part of magnesium sulfate heptahydrate, 2 parts of potassium dihydrogen phosphate, 0.025 part of vitamin B and 1000 parts of water.
Wheat medium: 15g wheat and 22.5mL liquid modified PD medium.
1.1.3 major instruments
Constant temperature incubator (Shanghai-constant scientific instruments Co., ltd.); VEX-600 digital microscope (Osaka, crohn's, japan); medical refrigerator (sea) at 4 ℃; -20 ℃ freezer refrigerator (hal); ultra-low temperature preservation boxes (hal); vacuum freeze dryer (LABCONCO).
1.2 test methods
1.2.1 methods of preparation and preservation of conidia
The strain is inoculated on a solid modified potato glucose culture medium, and is cultured for 3 days under the dark condition at the temperature of 23 ℃ and then is transferred to the light condition for culture. When mycelium grows to contact the inner wall of the culture dish, mycelium blocks (diameter 3-5 cm) or spores (1×10) at the edge of colony are taken 6 Individual spores/mL) was transferred to 20mL of liquid modified potato medium and placed in a shaker at 23℃and shake-cultured at 150rpm for 3d. After shaking culture, diluting the bacterial suspension (20-60 mycelium pellets/mL) after shaking culture with sterile distilled water for 2-5 times to prepare bacterial suspension, wherein the final concentration is 10-12 mycelium pellets/mL. And 5mL of the diluted bacterial suspension is inoculated into a sterilized wheat culture medium, and is cultured for 3d under the dark condition at 23 ℃ and then is cultured for 17d by using visible light. The spores of the cicada fungus cultivated for 20d are filtered and collected by two layers of sterile gauze in an ultra-clean workbench, packaged into a 1.5mL sterilizing centrifuge tube, sealed by a sealing film and respectively stored in a refrigerator at-80 ℃, at-20 ℃, at 4 ℃ and in a constant temperature incubator at 23 ℃ and at 37 ℃.
1.2.2 optimization of preservation temperature and period
Standing and preserving spore powder of Isaria cicadae stored in incubator at-80deg.C, -20deg.C, 4 deg.C and 23 deg.C and 37 deg.C for 0.5 month, 1 month, 3 months and 6 months respectively (whereinThe strain was further preserved for 12 months and 24 months under explicitly preferred preservation conditions). After a predetermined storage time has been reached, 1mL ddH is added 2 O and 0.05% Tween-80. In order to fully break up the spore powder, the centrifuge tube is put on an oscillator to oscillate for 1 minute, and after the conidium is completely broken up uniformly, the 1X 10 is prepared 6 Individual spores/mL spore suspension. A5 mm culture medium hole is drilled out in the center of the improved PDA culture medium by a puncher, 10 mu L of spore suspension is inoculated in the center of the PDA culture medium and placed in a constant temperature incubator at 23 ℃ for culture, the colony diameter is measured by a crisscross method, and the hypha growth condition is measured. Subsequently, the spore suspension was mixed with the modified PD medium 1:1 and placed on a biconcave slide, and placed in a constant temperature incubator at 23℃for 16h, and the spore germination of the strain was microscopically observed, and biological and technical replicates were performed 3 times.
In addition, mycelium pellets (3-5 cm in diameter, spore free) were placed in 1.5mL centrifuge tubes and stored in a-80 ℃, -20 ℃,4 ℃ refrigerator and 23 ℃ incubator for 3 days, 7 days and 14 days, respectively (wherein, after the preservation effect was confirmed, it was further determined whether to be used for the subsequent preservation). After a predetermined preservation time is reached, the state of the preserved strain (for example, the water accumulation of a centrifuge tube indicates that the water content of the culture medium is reduced, the strain is immersed in water and is in an anaerobic state) is observed, mycelium blocks are inoculated in the center of the modified PDA culture medium, the culture is carried out in a constant temperature incubator at 23 ℃, the colony diameter is measured by adopting a crisscross method, the mycelium growth condition is measured, and 3 biological and technical repetitions are carried out.
1.2.3 Effect of storage conditions on mycelium biomass
Standing and preserving in a constant temperature incubator of-80deg.C, -20deg.C, 4 deg.C and 23 deg.C for 6 months, adding 1mL ddH respectively 2 Placing the centrifuge tube on an oscillator, oscillating for 1min, and completely dispersing to obtain 1×10 powder 6 Individual spores/mL spore suspension. 200. Mu.L of the spore suspension was pipetted into modified PD liquid medium at 23℃at 150rpm and shake-cultivated in the dark for 4d. Washing mycelium with the obtained bacterial suspension, weighing mycelium biomass, and freeze-drying in vacuum freeze dryerThe mycelium biomass was measured and 3 biological and technical replicates were performed.
1.2.4 detection of infection Capacity
In reference to earlier studies due to the limitations of current laboratory raising cicada, the present application utilizes pupae of tussah (Antheraea pernyi) to inoculate conidium of Isaria cicadae. 1mL ddH was added to spore powder stored for 6 months, 12 months and 24 months 2 O and 0.05% Tween-80, oscillating to obtain 1×10 6 Individual spores/mL spore suspension. 1mL of spore suspension is sucked and added on sterile paper, the surface of the tussah pupa body is covered with the sterile paper, the light culture is carried out after the dark culture is carried out for 3d, the sterile paper is removed after the culture is carried out for 8d, the sterile water is supplemented in time, and the growth condition is observed after 30 d.
2. Results and analysis
2.1 influence of stored mycelium blocks on mycelium growth
The storage state of the hyphae and the growth condition of the activated hyphae are observed by storing the hyphae in a mode of hyphae blocks at different temperatures for a certain period of time. The storage evaluation result shows that after the strains are preserved for 7 days and 14 days, water accumulation occurs in the centrifuge tube at the temperature of minus 80 ℃ and minus 20 ℃, which indicates that the water content of the strain culture medium is reduced, and the strains are easily polluted by bacteria so as to influence the activity of the strains. Wherein the water content of the culture medium is reduced by 3.6% and 6.9% after 7 days and 14 days of preservation period at-80 ℃; whereas the water content of the medium at-20℃was reduced by 9.8% and 19.6% after 7 and 14 days of the storage period (FIG. 11). However, no water accumulation phenomenon is seen in the centrifuge tube after the spore powder is preserved for 24 months at different temperatures, which indicates that the strain preservation state is stable.
After preservation as a mycelium pellet at different temperatures, the mycelium growth was observed to change. The result shows that: after 3, 7 and 14 days of storage period, the strain stored in the form of mycelium block is activated, and the mycelium grows slowly compared with the spore powder. Wherein, after only 14 days of preservation period, the mycelium growth retardation was preserved at different temperatures (-80 ℃, -20 ℃,4 ℃ and 23 ℃) by 10.0%, 22.7%, 9.5% and 9.3%, respectively (fig. 12). Therefore, based on the phenomenon that the water content of the culture medium is reduced and the growth vigor of hyphae is reduced at different temperatures after a period of 14 days of short-term preservation, the corynespora cicadae mainly adopts a spore powder mode to carry out subsequent preservation condition optimization and preservation effect evaluation.
2.2 storing spore hypha growth
As a result of evaluating the growth condition of spore hyphae, it was found that the spore powder showed a change in hyphae growth after storage at different temperatures. The mycelium diameter was the largest when stored for 3 months at different temperatures, indicating that spores were most active when stored for 3 months. And no longer grows when stored at 23 ℃ for 6 months, room temperature is not suitable for preserving spore powder. The colony diameters of spore powder stored at 4℃were significantly higher than other temperatures, -80℃was next to 4 ℃. Although (ultra) cryopreservation is commonly used as an ambient temperature condition for strain preservation, isaria cicadae has better preservation at 4℃and-20℃and-80℃can also be used to preserve spore powder (FIG. 1). From the results of 6 months, it can be seen that the colony diameters of the stored spore powder at 4℃and-80℃are significantly higher than other temperatures, and thus further preserved. The colony diameters of spores were not greatly different from 6 months after 12 months and 24 months of storage at 4℃and-80℃respectively (FIG. 2).
2.3 spore germination of reservoir spores
By evaluating the germination situation of the stored spores, the spore powder is stored at different temperatures, and the spore powder is taken out after a preset preservation time to observe that the germination situation of the spores can be changed greatly. The germination rate of spores stored at 4 ℃ is up to 70-80% within the range of 6 months, and the germination rate is obviously higher than other temperatures; the spore germination rate stored at-80 ℃ is not high, but is relatively stable, and the spore germination rate is kept between 30 and 40 percent; spore germination rate was consistently reduced from 37% to 7% when stored at-20 ℃; spores stored at 23 ℃ maintained germination rate above 40% for 3 months, and no longer germinated for 6 months, and therefore were not suitable for long-term storage (fig. 3). From the results of 6 months, it can be seen that 4℃and-80℃are more suitable for long-term preservation of spore powder, and thus further preservation. Spores stored at 4℃showed a significant change in spore germination rate of 56% after 12 months of storage and 10% for two years of storage. The germination rate of spores stored at-80 ℃ was 24% after 12 months of storage and 9% after two years, also changed significantly. Spore germination rates were substantially consistent for 24 months of storage at-80 ℃ and 4 ℃ (figure 4).
2.4 reservoir spore hypha biomass changes
The results of the evaluation of different preservation conditions show that the capability of the spore powder for forming mycelium pellets by shaking culture has obvious difference after being stored under different temperature conditions. Shaking spores stored at different temperatures for 6 months showed that spores stored at 23℃could no longer form mycelium pellets, but spores at-80 ℃, -20℃and 4℃were still able to form, and the number of mycelium pellets produced at 4℃was more than-80℃and-20 ℃ (FIG. 5). The mycelium biomass was compared and found to be significantly higher for sporulation stored at 4℃than for the other two temperatures (FIG. 6).
2.5 reservoir spore infectivity
The results of the evaluation of different preservation conditions show that after the spore powder is stored under different temperature conditions, the capability of infecting tussah silkworm chrysalis also has obvious difference (figure 7). Spores stored at different temperatures for 6 months were prepared as spore suspensions, and the results showed that spores stored at-80 ℃, -20 ℃,4 ℃ and 23 ℃ were all capable of infecting, whereas spores at 37 ℃ were no longer capable of infecting (fig. 8). The spore infectivity at-80 ℃ and 4 ℃ can reach 100 percent, the spore infectivity at-20 ℃ is 91.7 percent, and the spore infectivity at 23 ℃ is only 16.7 percent. Further preservation of spore powder at-80 ℃ and 4 ℃ showed that spores still had infectivity after 12 months and 24 months of storage (fig. 9), and the infectivity could reach 100% (fig. 10).
The above embodiments are only illustrative of the preferred embodiments of the present application and are not intended to limit the scope of the present application, and various modifications and improvements made by those skilled in the art to the technical solutions of the present application should fall within the protection scope defined by the claims of the present application without departing from the design spirit of the present application.
Claims (5)
1. A preservation method of Isaria cicadae spore powder, which is characterized by comprising the following steps:
obtaining spore powder of the Isaria cicadae, and hermetically preserving the Isaria cicadae in the form of spore powder at the ultralow temperature of-80 ℃ or the low temperature of 4 ℃;
the method for obtaining the spore powder of the corynespora cicadae comprises the following steps:
(1) Inoculating the Isaria cicadae on a solid modified potato glucose culture medium, and culturing until hyphae grow out; the solid modified potato dextrose culture medium comprises the following components in parts by weight: 200 parts of potato, 20 parts of glucose, 3 parts of peptone, 1 part of magnesium sulfate heptahydrate, 2 parts of potassium dihydrogen phosphate, 0.025 part of vitamin B,16 parts of agar and 1000 parts of water;
(2) Transferring the mycelium and the spores to a liquid modified potato dextrose medium, and culturing to obtain a bacterial suspension; the liquid modified potato glucose culture medium is the solid modified potato glucose culture medium, and agar is not added in the solid modified potato glucose culture medium;
(3) Inoculating the bacterial suspension to a wheat culture medium, and culturing to obtain spore powder of the corynespora cicadae; the wheat culture medium comprises the following components in parts by weight: 15 parts of wheat and 22.5 parts of liquid modified potato dextrose medium.
2. The preservation method according to claim 1, wherein in the step (1), after culturing at 23℃in the dark for 3 days, culturing is continued under light until the mycelium grows in contact with the inner wall of the culture vessel.
3. The method according to claim 1, wherein in the step (2), the culture is carried out at 23℃and 150rpm for 3 days to obtain a bacterial suspension.
4. The method according to claim 1, wherein in the step (3), the culture condition is that the obtained bacterial suspension is inoculated in the wheat culture medium according to an inoculum size of 5%, and after the dark culture at 23 ℃ for 3d, the culture is further carried out by light irradiation for 17d, and then spore powder of Isaria cicadae is collected.
5. Use of a preservation method according to any one of claims 1-4 for preventing degradation of corynespora cicadae.
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| CN108901587A (en) * | 2017-04-18 | 2018-11-30 | 浙江泛亚生物医药股份有限公司 | A kind of solid culture method of cicada fungus |
| CN112913585A (en) * | 2021-02-02 | 2021-06-08 | 河北民族师范学院 | Preparation method of cordyceps sobolifera mycelium nutriment |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103571756A (en) * | 2012-07-30 | 2014-02-12 | 浙江泛亚生物医药股份有限公司 | Test tube screening method of isaria cicadae strain and culture medium |
| CN107409742A (en) * | 2016-05-23 | 2017-12-01 | 浙江泛亚生物医药股份有限公司 | A kind of artificial culture method of cicada fungus |
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