CN114214199A - Preservation method of isaria cicadae spore powder - Google Patents
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Abstract
The invention 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 Isaria cicadae, and hermetically storing Isaria cicadae at ultralow temperature of-80 ℃ or low temperature of 4 ℃ in the form of spore powder. The invention preserves isaria cicadae in the form of spore powder, the spore powder is placed in different temperature environments, the preservation conditions such as time period and the like are detected, and the standards of hypha growth, spore germination, mycelium biomass in bacterial suspension, infection capability of the mycelium biomass on tussah pupa and the like are combined for evaluation, so that the spore powder is finally preserved at the ultralow temperature of-80 ℃ or the low temperature of 4 ℃, the activity can be kept for more than 24 months, and the method is an optimal scheme for long-term preservation, and has important significance for filling the technical blank in the field of strain preservation of isaria cicadae and providing high-quality and stable isaria cicadae germplasm resources.
Description
Technical Field
The invention 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 Ascomycota, Chaetomium, Hypocreales, Cordyceps, Isaria. From ancient times to present, most medical classics are widely acknowledged only with the name of cordyceps sobolifera, and the cordyceps sinensis can accurately and vividly summarize the characteristics of the cordyceps sinensis, is a traditional precious Chinese medicinal material in China and has a long use history. Compared with the growth environment of cordyceps sinensis, isaria cicadae is widely distributed and mainly appears in the south of the Yangtze river of China, including Sichuan, Jiangsu, Zhejiang, Fujian, Anhui, Guangdong, Yunnan provinces and the like. Wild and artificially cultured Isaria cicadae have abundant nutritional components, and many active components beneficial to human health such as amino acids, adenosine, ergosterol, cordycepin and the like can be separated and extracted from Isaria cicadae.
Researches on artificial culture technology, biocontrol effect and the like of isaria cicadae have been widely carried out. However, in the production practice, a general problem encountered by cultivation technicians is that the artificially isolated strains are easy to have variation of production traits on a culture medium, namely, the phenomenon of strain degeneration, the infection capability of the degenerated strains is reduced, and isaria cicadae has the advantages of high safety and large spore yield, is a broad-spectrum entomogenous fungus with high pathogenicity on pests, and is very suitable for being used as an insecticide to control harmful insects. Therefore, it is important to effectively store the spores and improve the infection capacity of the spores by a preservation method.
Disclosure of Invention
The invention aims to provide a method for preserving spore powder of isaria cicadae, which aims to solve the problems in the prior art, and the spore powder is used for preserving the strain of isaria cicadae, so that the activity of the spore can be kept for more than 24 months under the refrigeration conditions of-80 ℃ and 4 ℃, the spore has the capability of infecting insects, and the method is a high-quality preservation method suitable for preserving the isaria cicadae strain for a long time.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a preservation method of isaria cicadae spore powder, which comprises the following steps:
obtaining spore powder of Isaria cicadae, and hermetically storing Isaria cicadae at ultralow temperature of-80 ℃ or low temperature of 4 ℃ in the form of spore powder.
Preferably, the method for obtaining spore powder of Isaria cicadae comprises the following steps:
(1) inoculating the isaria cicadae on a solid modified potato glucose culture medium, and culturing until hypha grows out; the solid modified potato glucose 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 monopotassium phosphate, 0.025 part of vitamin B, 16 parts of agar and 1000 parts of water;
(2) transferring the hyphae and the spores to a liquid improved potato glucose culture medium, and culturing to obtain a bacterial suspension; the liquid modified potato glucose culture medium is the solid modified potato glucose culture medium without agar;
(3) inoculating the bacterial suspension to a wheat culture medium, and culturing to obtain spore powder of isaria 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 glucose culture medium.
Preferably, in step (1), after culturing at 23 ℃ in the dark for 3d, the culture is continued under the illumination condition until the hyphae grow to contact the inner wall of the culture vessel.
Preferably, in step (2), the bacterial suspension is obtained by culturing at 23 ℃ and 150rpm for 3 d.
Preferably, in the step (3), the culture condition is that the obtained bacterial suspension is inoculated to the wheat culture medium according to the inoculation amount of 5 percent, after dark culture at 23 ℃ for 3 days, light culture is carried out for 17 days, and then spore powder of isaria cicadae is collected.
The invention also provides application of the preservation method in preventing isaria cicadae degeneration.
The invention discloses the following technical effects:
according to the invention, spore powder is placed in different temperature environments, preservation conditions such as time period and the like are detected, and the standards of hypha growth condition, spore germination condition, mycelium biomass in bacterial suspension, the infection capability of the mycelium biomass on tussah pupa and the like are combined for evaluation so as to establish an optimal preservation scheme and draw the following conclusion: the Isaria cicadae strain is preserved in the form of spore powder, and can be preserved for a short time at room temperature, and the preservation period can reach 3 months; the spore can still keep activity when being preserved for 6 months under the refrigeration condition of-20 ℃; and the spores can keep activity for more than 24 months under refrigeration conditions of-80 ℃ and 4 ℃, have the capability of infecting insects, and are the optimal temperature for long-term storage. The invention mainly aims at the technical blank in the field of Isaria cicadae Miq strain preservation, provides a high-quality preservation method for Isaria cicadae Miq strain, and provides a new method for ensuring high-quality and stable Isaria cicadae Miq germ plasm resources.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings 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 invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 shows the growth of spores stored at different temperatures for different storage periods;
FIG. 2 shows the growth of spores at different storage periods;
FIG. 3 shows spore germination of spores stored at different temperatures for different storage periods;
FIG. 4 shows spore germination at different storage periods for spores with suitable storage temperatures;
FIG. 5 shows hypha formation of Isaria cicadae spore powder stored at different temperatures for 6 months;
FIG. 6 shows the effect of Isaria cicadae spore powder stored at different temperatures for 6 months on hyphal biomass;
FIG. 7 shows that the Isaria cicadae spore powder stored at different temperatures for 6 months infects Antheraea pernyi pupae;
FIG. 8 is the effect of Isaria cicadae spore powder stored at different temperatures for 6 months on the infection activity of tussah pupa;
FIG. 9 shows the situation that the isaria cicadae spore powder is infected with the tussah pupa when stored for a long time at-80 ℃ and 4 ℃;
FIG. 10 shows the effect of Isaria cicadae spore powder on the infection activity of Antheraea pernyi pupa when stored at-80 deg.C and 4 deg.C for a long period of time;
FIG. 11 is a graph showing the effect of culture at different temperatures for 7 days and 14 days on the water content of the medium;
FIG. 12 is a graph showing the effect on hyphal growth of 3, 7 and 14 days of storage at different temperatures.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
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 invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, 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 invention 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 invention. All documents mentioned in this specification are incorporated by reference herein 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 present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
Example 1
Experimental materials and methods
1.1 test materials
1.1.1 test strains
The test strains were isolated and purified from the sporophore of Fujian Isaria cicadae and deposited in the mycology research laboratory of the plant protection institute of Shenyang university of agriculture.
1.1.2 test Medium
Solid modified potato glucose 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 glucose 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 culture medium: 15g wheat and 22.5mL liquid modified PD medium.
1.1.3 Main Instrument
Constant temperature incubator (shanghai-chang scientific instruments ltd); VEX-600 digital microscope (osaka, keyence, japan); medical refrigerator (hel) at 4 ℃; -20 ℃ freezer refrigerator (hel); ultra low temperature storage cases (hel); vacuum freeze drier (LABCONCO).
1.2 test methods
1.2.1 methods for preparation and preservation of conidia
Inoculating the strain to a solid modified potato glucose culture medium, culturing for 3d at 23 ℃ in the dark, and then culturing under the illumination condition. When hyphae grow and contact the inner wall of the culture dish, taking colony edgesMarginal hypha block (diameter 3-5cm) or spore (1X 10)6spores/mL) was transferred to 20mL liquid modified potato medium and placed in a shaker at 23 ℃ and shake cultured at 150rpm for 3 days. After the shaking culture is finished, diluting the bacterial suspension (20-60 mycelium pellets/mL) after the shaking culture by 2-5 times with sterile distilled water to prepare the bacterial suspension, wherein the final concentration is 10-12 mycelium pellets/mL. Inoculating 5mL of the diluted bacterial suspension into a sterilized wheat culture medium, culturing for 3d at 23 ℃ in the dark, and then culturing for 17d in the visible light. Filtering and collecting spores of Isaria cicadae cultured for 20 days in an ultraclean workbench by using two layers of sterile gauze, subpackaging into 1.5mL sterile centrifuge tubes, sealing by using a sealing film, and respectively storing in a refrigerator at minus 80 ℃, 20 ℃, 4 ℃ and constant temperature incubators at 23 ℃ and 37 ℃.
1.2.2 optimization of storage temperature and period
The spore powder of Isaria cicadae, which is stored in a refrigerator at-80 deg.C, -20 deg.C, 4 deg.C and constant temperature incubator at 23 deg.C and 37 deg.C, is kept still for 0.5 month, 1 month, 3 months, and 6 months, respectively (wherein, under the clearly preferred storage condition, the strain is further preserved for 12 months and 24 months). After the predetermined storage time was reached, 1mL ddH was added2O and 0.05% tween-80. To fully break up spore powder, the centrifuge tube is put on an oscillator to oscillate for 1 minute, and after the conidia are completely and uniformly broken up, the conidia are prepared into 1 × 106spores/mL spore suspension. Punching a 5mm culture medium hole in the center of the improved PDA culture medium by using a puncher, inoculating 10 mu L of spore suspension to the center of the PDA culture medium, culturing in a constant-temperature incubator at 23 ℃, measuring the diameter of a bacterial colony by adopting a cross method, and measuring the growth condition of the hyphae. Subsequently, the spore suspension and the improved PD medium are mixed in a ratio of 1:1 and then placed on a double-concave glass slide, the mixture is placed in a constant-temperature incubator at 23 ℃ for culturing for 16h, the spore germination condition of the strain is observed microscopically, and 3 times of biological and technical repetition are carried out.
In addition, the mycelium pellets (3-5 cm in diameter, no spores) were placed in a 1.5mL centrifuge tube, stored at-80 ℃, -20 ℃, 4 ℃ refrigerator and 23 ℃ incubator, and left for 3 days, 7 days and 14 days, respectively (wherein, after the preservation effect was confirmed, it was further determined whether to use for the subsequent preservation). After the preset preservation time is reached, observing the state of the preserved strains (if the water content of the culture medium is reduced as indicated by the water accumulation of a centrifuge tube, the strains are in an anaerobic state of soaking in water), inoculating a hypha block in the center of the improved PDA culture medium, culturing in a constant-temperature incubator at the temperature of 23 ℃, measuring the diameter of a bacterial colony by adopting a cross method, measuring the growth condition of the hypha, and repeating the biology and the technology for 3 times.
1.2.3 Effect of storage conditions on hyphal Biomass
Adding 1mL ddH into Isaria cicadae spore powder stored at-80 deg.C, -20 deg.C, 4 deg.C in refrigerator and 23 deg.C constant temperature incubator for 6 months2O and 0.05% Tween-80, placing the centrifuge tube on an oscillator, oscillating for 1min, and preparing into 1 × 10 powder after it is completely dispersed6spores/mL spore suspension. 200. mu.L of spore suspension was pipetted into modified PD broth and incubated at 23 ℃ and 150rpm in the dark for 4 days. Washing mycelia from the obtained bacterial suspension, weighing mycelia biomass, freeze-drying in a vacuum freeze-drying machine, and repeating the biological and technical processes for 3 times.
1.2.4 detection of infectivity
In view of the limitation of the current laboratory feeding of cicadas, the invention utilizes pupa of tussah (Antheraea pernyi) to inoculate conidia of Isaria cicadae. Adding 1mL ddH into spore powder preserved for 6 months, 12 months and 24 months2O and 0.05% Tween-80, and shaking to obtain 1 × 10 mixture6spores/mL spore suspension. Absorbing 1mL of spore suspension, adding the spore suspension onto sterile paper, covering the sterile paper on the surface of the tussah pupa, culturing in dark for 3d, then culturing in light, removing the sterile paper after culturing for 8d, supplementing sterile water on time, and observing the growth condition after 30 d.
Second, results and analysis
2.1 storage of hyphal clumps affects hyphal growth
The storage state of the mycelia and the growth of the activated mycelia were observed by storing the mycelia in a pellet form at different temperatures for a certain period of time. Storage evaluation results show that after the strains are stored for 7 days and 14 days, water is accumulated in the centrifugal tube at the temperature of minus 80 ℃ and minus 20 ℃, which indicates that the water content of the strain culture medium is reduced, so that the strains are easily polluted by bacteria to further influence the strain activity. Wherein, after the water content of the culture medium is preserved for 7 days and 14 days at the temperature of minus 80 ℃, the water content of the culture medium is reduced by 3.6 percent and 6.9 percent; whereas at-20 ℃ the water content of the medium decreased by 9.8% and 19.6% after 7 and 14 days storage cycles (FIG. 11). However, after the spore powder form is preserved for 24 months at different temperatures, no water accumulation phenomenon appears in the centrifuge tube, which indicates that the strain preservation state is stable.
After the culture medium was stored in the form of a hypha block at different temperatures, the change in hyphal growth was observed. As a result, it was found that: after 3 days, 7 days and 14 days of preservation period at different temperatures, the hypha growth is overall slow compared with that of the hypha powder after the strain preserved in the hypha block form is activated. Wherein hypha growth was slowed by 10.0%, 22.7%, 9.5% and 9.3% respectively after only 14 days of storage period at different temperatures (-80 deg.C, -20 deg.C, 4 deg.C and 23 deg.C) (FIG. 12). Therefore, the isaria cicadae will be subjected to subsequent preservation condition optimization and preservation effect evaluation mainly in a spore powder mode based on the phenomena of water content reduction of the culture medium at different temperatures and hypha growth activity reduction after the short-term preservation for 14 days.
2.2 storage of spore hyphal growth
The evaluation of the result of the hypha growth of the stored spores shows that the hypha growth of the spore powder changes after the spore powder is stored at different temperatures. At different temperatures, the hyphal diameter was maximal during 3 months of storage, indicating that the activity was highest for 3 months of storage of the spores. And no longer grows after 6 months of storage at 23 ℃, so that the room temperature is not suitable for storing the spore powder. The colony diameter stored at 4 ℃ spore powder is significantly higher than other temperatures-80 ℃ is second only to 4 ℃. Although the (ultra) cryopreservation is commonly used as the environmental temperature condition for strain preservation, Isaria cicadae can be preserved better at 4 deg.C, and-20 deg.C and-80 deg.C can also be used for preserving spore powder (FIG. 1). As can be seen from the results of 6 months, the colony diameters of the spore powder stored at 4 ℃ and-80 ℃ were significantly higher than those at other temperatures, and thus further preserved. The colony diameters of spores were not much different from 6 months after 12 months and 24 months of storage at 4 ℃ and-80 ℃ (fig. 2).
2.3 storage of spore germination
As a result of the evaluation of the germination of the stored spores, it was found that when the spore powder was stored at different temperatures, it was taken out and observed that the germination of the spores was greatly changed after a predetermined preservation time. The spore germination rate of the spores stored at 4 ℃ is up to 70-80% within the range of 6 months of storage, and is obviously higher than that of other temperatures; the germination rate of the spores stored at-80 ℃ is not high, but is relatively stable and is kept between 30 and 40 percent; the spore germination rate of the spore stored at the temperature of 20 ℃ below zero is reduced all the time, and is reduced from 37 percent to 7 percent; spores stored at 23 ℃ maintained their germination rate above 40% for 3 months and did not germinate for 6 months, and thus 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 therefore further preservation. Spores stored at 4 ℃ showed a spore germination rate of 56% after 12 months of storage and 10% after two years of storage, which was significantly changed. The germination rate of spores stored at-80 ℃ was 24% after 12 months of storage and 9% after two years, and also changed significantly. The spore germination rates were essentially consistent when stored at-80 ℃ and 4 ℃ for 24 months (FIG. 4).
2.4 storage of spore hyphal Biomass Change
Results of different preservation conditions are evaluated to find that the spore powder has obvious difference in the mycelium pellet forming capability by shaking culture after being stored at different temperatures. When spores stored at different temperatures for 6 months were shaken, it was shown that spores stored at 23 ℃ could no longer form mycelial pellets, while spores at-80 ℃, -20 ℃ and 4 ℃ could still form, and the number of mycelial pellets produced at 4 ℃ was more than-80 ℃ and-20 ℃ (FIG. 5). Comparison of the hyphal biomass revealed that the sporulated hyphal biomass stored at 4 ℃ was significantly higher than the other two temperatures (FIG. 6).
2.5 spore infectivity storage
Evaluation of results of different preservation conditions shows that the capacity of infecting tussah pupae of the spore powder after being stored under different temperature conditions is obviously different (figure 7). Spores stored at different temperatures for 6 months were made into spore suspensions to infect tussah pupae, and the results showed that spores stored at-80 deg.C, -20 deg.C, 4 deg.C and 23 deg.C all had the infecting ability, while spores at 37 deg.C had no infecting ability (FIG. 8). The spore infectivity of the spore stored at-80 ℃ and 4 ℃ can reach 100 percent, the spore infectivity of the spore stored at-20 ℃ is 91.7 percent, and the spore infectivity of the spore stored at 23 ℃ is only 16.7 percent. Further preservation of spore powder at-80 ℃ and 4 ℃ showed that the spores were still infested after 12 months and 24 months of storage (FIG. 9), and the infesting capacity was 100% (FIG. 10).
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (6)
1. The preservation method of isaria cicadae spore powder is characterized by comprising the following steps of:
obtaining spore powder of Isaria cicadae, and hermetically storing Isaria cicadae at ultralow temperature of-80 ℃ or low temperature of 4 ℃ in the form of spore powder.
2. A preservation process according to claim 1, wherein the method of obtaining spore powder of isaria cicadae comprises the steps of:
(1) inoculating the isaria cicadae on a solid modified potato glucose culture medium, and culturing until hypha grows out; the solid modified potato glucose 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 monopotassium phosphate, 0.025 part of vitamin B, 16 parts of agar and 1000 parts of water;
(2) transferring the hyphae and the spores to a liquid improved potato glucose culture medium, and culturing to obtain a bacterial suspension; the liquid modified potato glucose culture medium is the solid modified potato glucose culture medium without agar;
(3) inoculating the bacterial suspension to a wheat culture medium, and culturing to obtain spore powder of isaria 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 glucose culture medium.
3. The preservation method according to claim 2, wherein in the step (1), after culturing at 23 ℃ in the dark for 3 days, the culture is continued under light conditions until hyphae grow to contact the inner wall of the culture vessel.
4. The preservation method according to claim 2, wherein in the step (2), the bacterial suspension is obtained by culturing the mixture at a temperature of 23 ℃ and a speed of 150rpm for 3 d.
5. The preservation method according to claim 2, wherein in the step (3), the culture conditions are that the obtained bacterial suspension is inoculated on the wheat culture medium according to the inoculation amount of 5%, after dark culture at 23 ℃ for 3d, illumination culture is carried out for 17d, and then spore powder of Isaria cicadae is collected.
6. Use of a preservation process according to any one of claims 1 to 5 for preventing degeneration of isaria cicadae.
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| 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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