Long-term preservation and revival method for parasitic fungi of algae

Abstract

The invention discloses a long-term storage and revival method for algae parasitic fungi, and relates to the technical field of storage of algae parasitic fungi. The preservation method of the algae parasitic fungi comprises the following steps: inoculating strains of the parasitic fungi into algae host cells, transferring after reaching a preset infection rate, subpackaging in a centrifugal tube container, standing for layering, and removing supernatant on the upper layer; and uniformly shaking the container containing the residual product, sealing, storing at 4 DEG C, and shaking the container every 6 months. The reviving method of the algae parasitic fungi comprises the following steps: taking out the container, uniformly shaking, sucking a sample, adding the sample into an algae culture, and culturing until the algae cell infection rate reaches 100%; and standing the culture system, and sucking the liquid in the middle layer in the container as a strain to complete the reactivation of the parasitic fungi. According to the method disclosed by the invention, the normal metabolic activity of the algae parasitic fungi can be effectively ensured within the storage period of 1-3 years; the revived bacteria can complete the complete infection of the algae host, and the infection efficiency is high.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of preservation of algal parasitic fungi, in particular to a long-term preservation and revival method of algal parasitic fungi. BACKGROUND

[0002] Algae are a class of photosynthetic protists, including single-celled and multicellular types. They have the characteristics of wide distribution, rapid growth, high photosynthetic efficiency, and rich in various high-value metabolites, and have been widely used in food, health products, and aquaculture feed. Haematococcus pluvialis is rich in active ingredients such as super antioxidant astaxanthin, which can be used in health food to play the role of antioxidant, immune enhancement, and relief of visual fatigue; in the field of cosmetics, it can play the role of anti-aging, sun protection, and skin repair; it also has important application value in biological medicine and feed additives. Chromochloris zofingiensis also has significant application prospects, it can grow heterotrophically and has high yield, and has broad industrialization potential in astaxanthin production field, and it can also efficiently remove antibiotics and other pollutants in wastewater, and the harvested algal cells contain various high-value bioactive substances. Scenedesmus dimorphus has outstanding sewage treatment capacity, including water purification, nitrogen and phosphorus removal, heavy metal removal, and rich in protein, carotenoids, oil, and other nutrients, making it an excellent feedstock or bioenergy development object, and showing significant application value.

[0003] However, in the cultivation process of Haematococcus pluvialis, Chromochloris zofingiensis, and Scenedesmus dimorphus, the problem of pathogenic microorganism infection is increasingly prominent. The invasion of various pathogenic microorganisms such as fungi, bacteria, and protozoa often leads to a decrease in the yield and quality of the above-mentioned algae, severely restricting the sustainable development of their product applications. For example, a parasitic fungus named Paraphysoderma sedebokerense (P. sedebokerense) can specifically infect Haematococcus pluvialis, Scenedesmus dimorphus, and Chromochloris zofingiensis, causing massive death of these algae cells and collapse of the culture system in a short time, which greatly limits the application process of the above-mentioned algae.

[0004] In order to reduce the economic losses caused by P. sedebokerense infection of algae, it is necessary to carry out in-depth research on its infection mechanism and develop anti-infection drugs or anti-infection production methods on this basis. Therefore, it is very important to establish a research model of P. sedebokerense and algae. Many research institutions at home and abroad have carried out related researches on the infection of P. sedebokerense to algae. However, the culture method of P. sedebokerense is complex, the infection period is short, and after infecting the algal host, its spores will be dormant or die. Each experiment needs to prepare fresh fungal inoculum with infection activity, which makes it very inconvenient to obtain infectious spores for subsequent research in time. Therefore, it is necessary to establish a set of strain preservation technology suitable for algal parasitic fungi to provide strain samples for a long time.

[0005] Currently, there have been some studies on the preservation method of P. sedebokerense, mainly including: (1) P. sedebokerense can be maintained by artificially preparing nutrient-rich liquid medium or solid medium. However, this method needs to be subcultured every 5-7 days, and needs to purchase a constant temperature incubator to maintain the stability of culture temperature, rotation speed, etc. The preservation time is short and time-consuming and labor-consuming. (2) P. sedebokerense relies on the algal host in the parasitic stage, and needs to be added to the cell culture medium containing the algal host to maintain its parasitic nutrition process, but this method also needs to be subcultured every 5-7 days, and always needs to add fresh algal host cells, which is time-consuming and labor-consuming. (3) P. sedebokerense is preserved by low-temperature freezing preservation technology. This method adds dimethyl sulfoxide (DMSO) as a cryoprotective liquid to the culture medium containing P. sedebokerense, then incubates the sample containing P. sedebokerense and cryoprotective liquid at room temperature for 10-15 min to allow DMSO to fully penetrate into the cells, then uses a controlled rate freezer to cool the sample to -40℃ at a rate of 1℃ / min and keep it for 15 min, and then put the sample into liquid nitrogen for long-term preservation.

[0006] According to the current reports, the low-temperature freezing preservation technology can maintain the infection activity of P.sedebokerense in a 13-day preservation process, and there is no longer preservation time of the strain preservation report. However, the method has many shortcomings, for example, the strain preservation process is complicated, the liquid nitrogen tank and the cooler and other instruments and equipment are very expensive, the operation process is complicated, skilled workers are needed to operate, and it is not conducive to the repeated strain preservation method. SUMMARY

[0007] The current algal parasitic fungus strain preservation technology has the problems of poor operation convenience, complicated process, many experimental steps, much preparation work, high equipment investment and daily maintenance cost, high technical requirements for technical personnel, poor stability of strain preservation effect, and short periodicity of algal parasitic fungus strain preservation. In order to solve these problems, the application provides a long-term preservation and revival method of algal parasitic fungus, which is simple in operation, low in instrument and equipment investment, good in preservation effect, long in preservation period, and high in infection efficiency of the revived strain. The method is realized through the following technologies.

[0008] The application provides a long-term preservation and revival method of algal parasitic fungus, which comprises a preservation method and a revival method.

[0009] The steps of the preservation method comprise the following steps of inoculating a parasitic fungus strain into algal host cells.

[0010] When the infection rate of the algal host cells is 30-80%, the algal host cells are transferred into a container (for example, a centrifuge tube) for static stratification, and the supernatant is removed.

[0011] The container containing the remaining product is shaken, sealed, preserved at 4-8 DEG C, and the container is shaken every 6 months.

[0012] The steps of the revival method comprise the following steps of:

[0013] The container containing the parasitic fungus is taken out, shaken, and the sample is taken and added to the algal culture for culture until the infection rate of the algal cells reaches 80-100%.

[0014] The culture system is static, the liquid in the middle layer of the container is taken, and the revival of the parasitic fungus is completed.

[0015] The algal host cells are Haematococcus pluvialis, Chromochloris zofingiensis or Scenedesmus dimorphus algal cells, and the parasitic fungus is Paraphysoderma sedebokerense.

[0016] In the preservation method, after the parasitic fungi are inoculated into the algal host cells and cultured, the fungi cells and the infected algal host cells are aggregated with each other due to the entanglement effect of the fungal hyphae and naturally settle at the bottom of the centrifuge tube. The progeny spores of the fungi with low resistance newly germinated from the fungal mother and the fragments of the algal host cells broken due to the fungal infection are left in the supernatant due to the low settlement coefficient. Therefore, most of the progeny spores of the fungi with low resistance and the cell fragments can be discarded by pouring off the supernatant, so that the fungal mother with high resistance is reserved. When the long-term preservation at 4-8°C is performed, the container contains a large amount of the fungal mother and the infected algal host cells, and the algal host cells can serve as the nutrient source of the fungal mother to maintain the slow growth of the parasitic fungi under the conditions of low oxygen content and low temperature.

[0017] In the reviving method, when the infection rate of the algal cells by the parasitic fungi reaches 100%, the fungal bodies and the entangled algal cells settle at the bottom of the centrifuge tube after standing, and the newly germinated progeny spores are left in the supernatant due to the low settlement coefficient. The middle liquid containing the newly germinated fungal spores with low resistance but strong infection activity and metabolic capacity is taken by using a pipette. Therefore, the fungal spores can be used as the fungal strain to inoculate into the algal culture system for subsequent researches, such as the infection mechanism of the fungi or the screening of anti-infection agents.

[0018] Further, in the preservation method, the inoculation amount of the parasitic fungi into the algal host cells is 0.1-10%.

[0019] Further, in the preservation method, the inoculation amount of the parasitic fungi into the algal host cells is 1%.

[0020] Further, in the preservation method, the volume ratio of the residual product to air in the container containing the residual product is 1:(1-10).

[0021] Further, in the preservation method, the volume ratio of the residual product to air in the container containing the residual product is 1:1.5.

[0022] Further, in the preservation method, the temperature for the sealed preservation is 4°C.

[0023] Further, in the reviving method, the formula of the algal culture is: KNO3 150-1500 mg, K2HPO4 20-40 mg, MgSO4·7H2O 75 mg, CaCl2·2H2O 36 mg, citric acid 6 mg, ferric-ammonium citrate 6 mg, EDTA-Na2 1 mg, Na2CO3 20 mg, H3BO3 2.86 mg, MnCl2·4H2O 1.81 mg, ZnSO4·7H2O 0.22 mg, CuSO4·5H2O 0.08 mg, Na2MoO4·2H2O 0.39 mg, Co(NO3)2·6H2O 0.05 mg.

[0024] Further, in the reviving method, the formula of the algal culture is: KNO3 1500 mg, K2HPO4 40 mg, MgSO4·7H2O 75 mg, CaCl2·2H2O 36 mg, citric acid 6 mg, ferric-ammonium citrate 6 mg, EDTA-Na2 1 mg, Na2CO3 20 mg, H3BO3 2.86 mg, MnCl2·4H2O 1.81 mg, ZnSO4·7H2O 0.22 mg, CuSO4·5H2O 0.08 mg, Na2MoO4·2H2O 0.39 mg, Co(NO3)2·6H2O 0.05 mg.

[0025] Further, in the reviving method, the volume ratio of the sample and the algal culture is 1: (10-1000).

[0026] Further, in the reviving method, the volume ratio of the sample and the algal culture is 1:10.

[0027] Compared with the prior art, the algae parasitic fungus long-term preservation reviving method has the advantages of:

[0028] 1. Simple operation: the algae parasitic fungus long-term preservation reviving method provided by the application has few experimental steps, and the technical personnel need to operate few links in each step, so that the operation process is simple and easy to master and repeat.

[0029] 2. Low investment in instruments and equipment: the algae parasitic fungus long-term preservation reviving method provided by the application can use general laboratory common instruments and equipment, such as a super-clean workbench, a pipettor, a centrifuge tube and a refrigerator, without the need to additionally purchase instruments and equipment, thereby saving investment; even if the above instruments and equipment are not available, the above instruments and equipment can be purchased at a relatively low budget.

[0030] 3. Good preservation effect: The long-term preservation and revival method of the algal parasitic fungi provided by the application has been repeatedly practiced and verified by the research team of the applicant, and the technical stability and preservation effect have good repeatability, and the cell activity of the preserved fungi can be maintained during the long-term preservation process of 1-3 years.

[0031] 4. Long preservation period: The algal parasitic fungi samples preserved by the method of the application are taken out every 1 year, and the cell activity of the preserved fungi is detected, and it is found that the metabolic activity of the preserved fungi is normal during the preservation period of 1-3 years; after revival, it can complete the complete infection of the algal host, and the infection efficiency is high. This shows that the method of the application can effectively realize the long-term preservation and effective revival of the algal parasitic fungi. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 The flowchart of the long-term preservation and revival method of the algal parasitic fungi provided for example 1.

[0033] Figure 2 The microscopic observation results of the algal host Chlorella and the parasitic fungi at each node in the long-term preservation and revival method of the algal parasitic fungi provided for example 1.

[0034] Figure 3 The microscopic observation results of Chlorella and parasitic fungi after being preserved for different times by using the long-term preservation and revival method of the algal parasitic fungi of example 1, and the results after being stained with specific fluorescent dye for labeling algal parasitic fungi.

[0035] Figure 4 The cell activity of the parasitic fungi after being preserved for different times by using the long-term preservation and revival method of the algal parasitic fungi of example 1. The cell activity of the fungi after being preserved for 1-3 years was quantitatively detected by using a flow cytometer. DETAILED DESCRIPTION

[0036] The technical solutions of the application will be described below in a clear and complete manner. Obviously, the described embodiments are only some of the embodiments of the application, not all. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the application.

[0037] The application is a set of long-term preservation and revival method of algal parasitic fungi summarized by the applicant based on experimental results from 2016 to now about ten years.

[0038] In the following examples and comparative examples, the algal host cell used is Haematococcus pluvialis, purchased from the Scandinavian Culture Center for Algae and Protozoa, University of Copenhagen, Denmark (SCCAP NO. K-0084). The parasitic fungus used is Paraphysoderma sedebokerense, isolated by the applicant from the infected liquid medium environment of a Haematococcus pluvialis cultivation site under natural conditions (GenBank ID MN203631). After extensive experimental research, it was found that the source and manner of isolation of Paraphysoderma sedebokerense had no effect on the long-term preservation and revival method thereof.

[0039] In some embodiments of the present application, the long-term preservation and revival method of the algal parasitic fungus comprises a preservation method and a revival method.

[0040] The preservation method comprises the following steps:

[0041] (1) inoculate the algal host cell with the spores of the parasitic fungus to start infecting the algal host cell;

[0042] (2) when the infection rate of the algal host cell is 30-80%, transfer it to a container (e.g. a centrifuge tube) for static stratification, and remove the supernatant;

[0043] (3) shake the container containing the remaining product, seal it, and store it at 4°C, and shake the container every 6 months;

[0044] The revival method comprises the following steps:

[0045] (1) take out the container containing the parasitic fungus, shake it, and add the sample to the algal culture for cultivation until the infection rate of the algal cells reaches 80-100%;

[0046] (2) let the cultivation system stand, and take the liquid in the middle layer of the container to complete the revival of the parasitic fungus.

[0047] Optionally, the algal host cell is an algal cell of Haematococcus pluvialis, Chromochloris zofingiensis or Scenedesmus dimorphus.

[0048] Optionally, the parasitic fungus is Paraphysoderma sedebokerense.

[0049] In some embodiments of the application, when the algal host cells are preserved, the parasitic fungus is inoculated into the algal host cells at an inoculation amount of 0.1-10%.

[0050] Optionally, the parasitic fungus is inoculated into the algal host cells at an inoculation amount of 1%.

[0051] In some embodiments of the application, when the algal host cells are preserved, the volume ratio of the residual product to air in the container containing the residual product is 1:(1-10).

[0052] Optionally, the volume ratio of the residual product to air in the container containing the residual product is 1:1.5.

[0053] Optionally, in the preservation method, the temperature for the sealed preservation is 4°C.

[0054] In other embodiments of the application, when the algal host cells are revived, the algal culture used can be any commonly used or commercially available algal culture medium.

[0055] Optionally, a commonly used medium for culturing green algae is used. For example, BG11 medium is used.

[0056] The formula of the algal culture used in the application is: KNO3 150-1500 mg, K2HPO4 20-40 mg, MgSO4·7H2O 75 mg, CaCl2·2H2O 36 mg, citric acid 6 mg, ferric-ammonium citrate 6 mg, EDTA-Na2 1 mg, Na2CO3 20 mg, H3BO3 2.86 mg, MnCl2·4H2O 1.81 mg, ZnSO4·7H2O 0.22 mg, CuSO4·5H2O 0.08 mg, Na2MoO4·2H2O 0.39 mg, Co(NO3)2·6H2O 0.05 mg.

[0057] When the algal host cells are revived, the volume ratio of the sample to the algal culture is 1:(10-1000).

[0058] Optionally, the volume ratio of the sample to the algal culture is 1:10.

[0059] Example 1

[0060] The long-term preservation and revival method of the algal parasitic fungus provided in this example is as follows:Figure 1 As shown, according to the preservation method and the revival method, the respective specific steps are as follows.

[0061] (1) Start the infection of the algal host cells by introducing 1 mL of the fungal species with an optical density value OD 5 = 0.03 into 100 mL of the algal host cells with a cell density of 4 x 10 600 = 0.03 into 100 mL of the algal host cells with a cell density of 4 x 10

[0062] (2) When the infection rate of the algal host cells is about 80%, take out 50 mL of the sample in a super-clean bench, and add it into a 50 mL sterile centrifuge tube and stand for about 5 min.

[0063] At this time, due to the entanglement effect of the fungal hyphae, the fungal cells and the infected algal host cells adhere to each other and naturally settle at the bottom of the centrifuge tube; the low-resistance daughter spores newly germinated from the fungal mother and the algal cell fragments broken due to fungal infection are left in the supernatant due to the low sedimentation coefficient.

[0064] (2) Slowly tilt the centrifuge tube, pour out the supernatant, and reserve about 20 mL of the lower sediment;

[0065] (3) Gently mix the lower sediment with the remaining culture solution, tighten the centrifuge tube cover, and seal it with a sealing film.

[0066] At this time, the centrifuge tube contains a large number of parasitic fungal spores and infected algal host cells, i.e., about 20 mL of the fungal sample and 30 mL of air in a 50 mL volume of the centrifuge tube.

[0067] (4) Place the centrifuge tube containing the fungal sample vertically in a 4°C refrigerator, and gently shake the centrifuge tube every about 6 months.

[0068] (5) Take out the preserved fungal sample centrifuge tube from the 4°C refrigerator, gently shake it in a super-clean bench, open the sealing film, and remove the centrifuge tube cover.

[0069] Use a pipette to take 10 mL of the preserved fungal sample and add it into the algal culture with a cell density of 4 x 10 5 = 0.03 into 100 mL of the algal host cells with a cell density of 4 x 10

[0070] The formula of the algal culture is: KNO3 1500 mg, K2HPO4 40 mg, MgSO4·7H2O 75 mg, CaCl2·2H2O 36 mg, citric acid 6 mg, ferric-ammonium citrate 6 mg, EDTA-Na2 1 mg, Na2CO320 mg, H3BO3 2.86 mg, MnCl2·4H2O 1.81 mg, ZnSO4·7H2O 0.22 mg, CuSO4·5H2O 0.08 mg, Na2MoO4·2H2O 0.39 mg, Co(NO3)2·6H2O 0.05 mg.

[0071] (6) After 5-7 days of culture, the infection rate of the parasitic fungi on the algal cells reaches more than 80%. Continue to culture until the infection rate reaches 100%.

[0072] (7) Take out 100 mL of the culture and pour it into two 50 mL centrifuge tubes, respectively, and stand for about 5 min.

[0073] At this time, the mycelium of the parasitic fungi and the algal cells wound by them settle at the bottom of the centrifuge tube, and the newly germinated daughter spores remain in the upper clear liquid due to the low settling coefficient.

[0074] Use a pipette to suck the liquid in the middle layer of the centrifuge tube. The newly germinated fungal spores contained in this layer of liquid have strong infection activity and metabolic capacity.

[0075] (8) The part of the newly germinated fungal spores can be used as a fungal strain and inoculated into the algal culture system for subsequent research on the infection mechanism of fungi or screening of anti-infection drugs and other experiments.

[0076] In the above-mentioned long-term preservation and revival process of the algal parasitic fungi, the microscopic observation results of the algal parasitic fungi are shown in FIG. 1. Figure 2 Among them, 1 is the microscopic observation result of the algal host cells selected; 2 is the microscopic observation result after the parasitic fungi are inoculated into the algal host cells and start to infect; 3 is the microscopic observation result when the infection rate reaches about 80%; 4 is the microscopic observation result when the parasitic fungi stored at 4°C are added to new algal host cells and revived to an infection rate of 100%; 5 is the microscopic observation result of the fungal spores released by the revived parasitic fungi, which are located in the middle layer of the centrifuge tube; 6 is the microscopic observation result of the parasitic fungal spores inoculated into the algal cells after the middle layer liquid is transferred out, which can be seen to have a strong infection effect.

[0077] To verify the preservation effect of the present method, we used a fluorescent dye BODIPY (Molecular Probes, USA) specially labeled the algal parasitic fungus P. sedebokerense to perform fluorescent staining on the preserved fungus samples stored for 1 year, 2 years, and 3 years, respectively; and used a fluorescence microscope to qualitatively observe the labeling condition, and used a flow cytometer to quantitatively detect the luminescence intensity of the preserved fungus to reflect the survival condition of the fungus. At the same time, it was compared with the newly prepared algal parasitic fungus infection sample to evaluate the influence of different preservation times on the activity of the algal parasitic fungus.

[0078] The test method for labeling the fungus with a fluorescent dye, and simultaneously detecting the survival condition of the fungus using a fluorescence microscope and a flow cytometer is as follows:

[0079] (1) 1 mL of sample was stained at room temperature in the dark for 10 min using a BODIPY dye solution with a final concentration of 50 μM at room temperature in the dark. Under this condition, since the algal cells were not stained by the BODIPY dye solution, the fungus could be stained, and the stronger the activity of the fungus cell, the higher the fluorescence brightness, so this method can be used to quantitatively count the number of active fungus.

[0080] (2) After the staining was completed, the sample was observed under a fluorescence microscope, the excitation light / emission light wavelength combination was adjusted to 488 / 525 nm, and the staining result was photographed.

[0081] At the same time, the relative fluorescence intensity of the fungus cell was quantitatively detected using a flow cytometer (Beckman Coulter, FC-500) to reflect the activity of the fungus cell after different preservation times. Three repeated samples were set for each time point to calculate the average fluorescence intensity, and the quantitative data was represented as mean ± S.D. on the column chart.

[0082] The results are shown in Figs. Figure 3 (Staining result) and 4 (column chart), it can be seen that the survival rate of the parasitic fungus (Paraphysoderma sedebokerense) gradually decreased with the prolongation of the preservation time. However, using the method of the present embodiment, the fungus samples preserved for 1-3 years all had very strong infection activity, and the preserved fungus samples could be successfully revived.

[0083] Example 2

[0084] In June 2020, the fungus sample preserved in September 2017 according to the method of Example 1 was taken out to carry out a revival experiment.

[0085] First, prepare Haematococcus pluvialis cells: use a 250 mL glass conical flask, add 100 mL of algae culture medium and algae seeds, and culture at 22°C until the algae cell density grows to about 4×10 5 Next, a fungal sample stored in November 2017 was taken, shaken well, and 10 mL was transferred to a conical flask. The sample was gently shaken and incubated in a constant-temperature shaker at 30°C and 100 rpm. After six days of incubation, samples were removed for microscopic examination and infection rate analysis, revealing that all algal cells were infected. This demonstrates that fungi stored for approximately three years using this method maintain good activity.

[0086] Example 3

[0087] In July 2025, the fungal sample preserved according to the method of Example 1 in July 2023 was taken out and a resurrection experiment was carried out.

[0088] First, prepare Haematococcus pluvialis cells: use a 250 mL glass conical flask, add 100 mL of algae culture medium and algae seeds, and culture at 22°C until the algae cell density grows to about 4×10 5 Next, a fungal sample stored in July 2023 was taken, shaken, and 1 mL was transferred to a conical flask. The sample was gently shaken and incubated in a constant-temperature shaker at 30°C and 100 rpm. After five days of incubation, samples were removed for microscopic examination and infection rate analysis, revealing that all algal cells were infected. This demonstrates that fungi stored for two years using this method maintain good activity.

[0089] In summary, using the algae-parasitic fungus (Paraphysoderma sedebokerense) as an example, the preservation method of the present invention, when used for one to three years and then revived according to the method of the present invention, can maintain good metabolic activity and effectively infect algal cells. Compared with the fungus preservation methods mentioned in existing literature (which have a shelf life of one week or 13 days), this shelf life is significantly extended.

[0090] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details of the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

Claims

1. A method for long-term preservation and revival of algae parasitic fungi, characterized in that: including preservation methods and resurrection methods; The steps of the preservation method include: inoculating parasitic fungi into algae host cells; When the infection rate of the algae host cells reaches 30-80%, the algae host cells are transferred to a container and allowed to stand for stratification, and the supernatant is removed; Shake the container containing the remaining product, seal it, store it at 4-8°C, and shake the container every 6 months; The steps of the resurrection method include: Taking out the container containing the parasitic fungus, shaking it well, taking a sample and adding it to the algae culture, and culturing it until the infection rate of the algae cells reaches 80-100%; The culture system is allowed to stand still, and the liquid in the middle layer of the container is sucked out to complete the revival of the parasitic fungus; The algae host cell is an algae cell of Haematococcus pluvialis, Chromochloris zofingiensis or Scenedesmus dimorphus; and the parasitic fungus is Paraphysoderma sedebokerense.

2. The long-term preservation and revival method of algae parasitic fungi according to claim 1, characterized in that: In the preservation method, the inoculation amount of the parasitic fungus inoculated into the algae host cells is 0.1-10%.

3. The long-term preservation and revival method of algae parasitic fungi according to claim 2, characterized in that: In the preservation method, the inoculation amount of the parasitic fungus inoculated into the algae host cells is 1%.

4. The long-term preservation and revival method of algae parasitic fungi according to claim 1, characterized in that: In the storage method, in the container containing the remaining product, the volume ratio of the remaining product to air is 1:(1-10).

5. The long-term preservation and revival method of algae parasitic fungi according to claim 4, characterized in that: In the storage method, in the container containing the remaining product, the volume ratio of the remaining product to air is 1:1.

5.

6. The long-term preservation and revival method of algae parasitic fungi according to claim 1, characterized in that: In the storage method, the sealed storage temperature is 4°C.

7. The long-term preservation and revival method of algae parasitic fungi according to claim 1, characterized in that: In the resurrection method, the formula of the algae culture is: KNO3 150-1500 mg, K2HPO4 20-40 mg, MgSO4·7H2O 75 mg, CaCl2·2H2O 36 mg, citric acid 6 mg, ferric-ammonium citrate 6 mg, EDTA-Na2 1 mg, Na2CO3 20 mg, H3BO3 2.86 mg, MnCl2·4H2O 1.81 mg, ZnSO4·7H2O 0.22 mg, CuSO4·5H2O 0.08 mg, Na2MoO4·2H2O 0.39 mg, and Co(NO3)2·6H2O 0.05 mg.

8. The long-term preservation and revival method of algae parasitic fungi according to claim 1, characterized in that: In the resurrection method, the formula of the algae culture is: KNO3 1500 mg, K2HPO4 40 mg, MgSO4·7H2O 75 mg, CaCl2·2H2O 36 mg, citric acid 6 mg, ferric-ammonium citrate 6 mg, EDTA-Na2 1 mg, Na2CO3 20 mg, H3BO3 2.86 mg, MnCl2·4H2O 1.81 mg, ZnSO4·7H2O 0.22 mg, CuSO4·5H2O0.08 mg, Na2MoO4·2H2O 0.39 mg, and Co(NO3)2·6H2O 0.05 mg.

9. The long-term preservation and revival method of algae parasitic fungi according to claim 1, characterized in that: In the resurrection method, the volume ratio of the sample to the algae culture is 1:(10-1000).

10. The long-term preservation and revival method of algae parasitic fungi according to claim 1, characterized in that: In the resurrection method, the volume ratio of the sample to the algae culture is 1:10.

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