A method of increasing the tolerance of corals to vibrio coralliiyticus

Abstract

The present application provides a method for improving the tolerance of corals to Vibrio coralliius, which comprises the following steps: first, culturing healthy Echinomorpha crassior and Acropora humilis; then, inoculating Vibrio coralliius to the two corals to screen out the coral, Acropora humilis, which is resistant to Vibrio coralliius and the coral, Echinomorpha crassior, which is susceptible to Vibrio coralliius; taking the screened Acropora humilis as a donor to culture microbial liquid; inoculating the microbial liquid to the screened Echinomorpha crassior, so as to improve the tolerance of the Echinomorpha crassior to Vibrio coralliius. The present application has the advantages of simple operation, safety and reliability, high efficiency of coral mixed liquid extraction and high microbial activity. The coral resistant to Vibrio coralliius is taken as a donor to prepare a mixed liquid, and the coral susceptible to Vibrio coralliius is treated to improve its tolerance. The method has low ecological side effects, can quickly and effectively improve the tolerance of the coral susceptible to Vibrio coralliius, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of bioengineering, and particularly relates to a method for improving the tolerance of corals to Vibrio corallilyticus. BACKGROUND

[0002] Coral reef ecosystem is an important part of marine ecosystem and one of the marine ecosystems with the highest primary productivity. Coral reef ecosystem has extremely high biodiversity and important research value. At present, coral reefs worldwide are continuously threatened by environmental changes such as global warming and ocean acidification. In addition, the outbreak of coral disease is an important inducement to aggravate the degradation of coral reefs. The outbreak of coral disease will lead to the loss of structure and function of coral reef ecosystem, causing irreversible damage to the coral reef ecosystem. The currently discovered coral diseases mainly include yellow band disease, white band disease, black band disease and bacterial bleaching, but less than ten pathogenic bacteria have been confirmed to cause coral disease, including Serratia marcescens 、 Vibrio tubiashii 、 Pseudoalteromonas pirati 、 Thalassotalea loyana 、 Vibrio shiloi and Vibrio corallilyticus Vibrio coralliilyticus . Different pathogenic bacteria infect different coral hosts and have different disease symptoms. However, the pathogenic mechanism of the remaining pathogenic bacteria except for Vibrio shiloi Vibrio shiloi is still to be explored. In order to prevent the damage of coral disease to coral reefs and the further degradation of coral reef ecosystem, many methods for coral reef restoration have been explored by the predecessors, and the methods for preventing and treating coral disease mainly include antibiotic therapy, phage therapy and probiotic therapy. The first two methods have the following problems:

[0003] 1. Antibiotic therapy has a significant effect on the prevention and treatment of coral disease, but it is easy to cause the generation of pathogen resistance and may lead to the accumulation of antibiotics in some organisms, which ultimately affects humans through food chain enrichment.

[0004] 2. Phage therapy has the advantages of high specific lysis and specificity, self-replication and environmental friendliness, but still has potential risks such as the generation of pathogen resistance and the transfer of virulence genes, and has less applicability.

[0005] The beneficial microorganisms used in probiotic therapy can be obtained from coral hosts and the environment, which has lower ecological side effects compared to other two methods of preventing and treating coral diseases, and has great practical potential and broad application prospects in the prevention and treatment of coral diseases. Since Leah Reshef proposed the "probiotic hypothesis" in 2006, many studies have further explored and extended the hypothesis. The experiments of coral beneficial microorganism transplantation based on related theories have achieved success in coral heat resistance, tolerance to petroleum pollutants and disease resistance, further confirming the feasibility of coral beneficial microorganism transplantation for maintaining coral health and the great application potential and broad prospects in the restoration of coral reef ecosystems. Probiotic transplantation has been deeply explored and has a relatively mature theoretical system in the treatment of human intestinal diseases, the prevention and treatment of crop diseases and other terrestrial microbial ecological communities, but its exploration in marine ecosystems such as coral reef ecosystems is still in its infancy. At present, the application of probiotic therapy in the prevention and treatment of coral diseases mainly depends on the screening and purification process of coral symbiotic probiotics and seawater probiotics, and single strain and multi-strain bacterial groups are often used in experiments. The experiments based on single strain or multi-strain bacterial groups mainly have the following problems:

[0006] 1. The screening, isolation and culture process of coral symbiotic beneficial microorganisms and seawater probiotics requires a long time.

[0007] 2. The stability of single strain or multi-strain bacterial groups obtained by isolation and culture in the new coral symbiotic microbial community is not as good as that of the complete original microbial community, and the probiotic effect may be discounted.

[0008] 3. Some coral probiotics may indirectly affect other microorganisms in the original coral symbiotic microbial community to exert probiotic effects, and single strain or multi-strain screening may miss such indirect beneficial probiotics.

[0009] 4. Whether single strain and multi-strain bacterial groups can stably exist in the new coral symbiotic microbial community still needs to be explored.

[0010] The related problems are key problems in the practice of coral beneficial microorganism transplantation, and effectively solving such bottleneck problems will help to improve the theory of coral beneficial microorganisms and promote the progress of coral disease prevention and treatment. Therefore, the present application has broad application prospects in the prevention and treatment of coral diseases, and is of great significance for the research and protection of coral reef ecosystems. SUMMARY

[0011] In view of the above problems existing in the coral probiotic transplantation work in the prior art, the present application provides a method for improving the tolerance of coral to Vibrio shiloi, which provides a new way for the research and protection of coral reef ecosystems.

[0012] The application is realized by the following technical scheme:

[0013] A method for improving the tolerance of corals to Vibrio corallilyticus, comprising the following steps:

[0014] S1: Select healthy Acropora cytherea and Montipora capitata in good growth state, and culture under the following growth conditions: temperature: 24-28℃, pH: 8.1-8.3, KH: 7.0±0.3, pH: 8.2±0.1, Ca 2+ : 400±15ppm, Mg 2+ : 1400±20ppm, PO4 3- <0.03ppm, NH 3+ <0.15ppm, NO 2- <0.1ppm, NO 3- ≈0ppm, and use halogen lamp to simulate natural light, and set the light duration: dark duration to 12h:12h;

[0015] S2: Rejuvenate Vibrio corallilyticus, centrifuge the bacterial liquid after 24h of culture at 8000rpm for 6min, remove the supernatant, then resuspend the precipitate in seawater to prepare Vibrio corallilyticus liquid;

[0016] S3: inoculate the Vibrio corallilyticus liquid into the Acropora cytherea and Montipora capitata obtained by culture in step S1 respectively, and culture for 11-24h, and select corals with tolerance and susceptibility to Vibrio corallilyticus;

[0017] S4: Take the Montipora capitata with tolerance to Vibrio corallilyticus selected in step S3 as a donor, wash the coral block with a sterile seawater by a dental cleaner to obtain a mixed liquid V1, and then measure the surface area S of the coral block with tin paper;

[0018] S5: filter out large particle impurities and coral mucus from the mixed liquid V1 with neutral filter paper to obtain a mixed liquid V2;

[0019] S6: centrifuge the mixed liquid V2 at 8000g for 5min, remove the supernatant, resuspend with sterile seawater, shake and mix to obtain a mixed liquid V3, i.e. the donor mixed liquid;

[0020] S7: inoculate the mixed solution V3 to the Pocillopora damicornis which is susceptible to V. corallivorus screened in step S3 as a test group, inoculate the Pocillopora damicornis which is susceptible to V. corallivorus screened in step S3 with the same amount of seawater as mixed solution V3 as a blank group; inoculate the Pocillopora damicornis which is susceptible to V. corallivorus screened in step S3 with mixed solution V3 and the same amount of V. corallivorus as a control group; and culture the Pocillopora damicornis in the test group, the blank group and the control group for 24-116 hours;

[0021] S8: measure the maximum quantum yield, zooxanthellae density, coral color index, total superoxide dismutase activity and catalase activity of the cultured Pocillopora damicornis, and evaluate the disease resistance effect by analyzing the difference in the occurrence of significant infection symptoms between the Pocillopora damicornis inoculated with and without mixed solution V3, and when the growth data of the Pocillopora damicornis inoculated with and without mixed solution V3 appear significant difference, it indicates that the mixed solution V3 can improve the tolerance of the susceptible Pocillopora damicornis to V. corallivorus.

[0022] The V. corallivorus used in the present application is a strain with a preservation number of MCCC 1A11086 from the Marine Microbial and Algal Culture Collection Center. Vibrio coralliilyticus

[0023] The symbiotic bacterial community of the Pocillopora damicornis has higher alpha diversity than the Pocillopora damicornis, and research shows that a highly diverse bacterial community indicates that the symbiotic bacterial community has greater plasticity to environmental stress, helps the host to adjust the microbial community structure to cope with environmental changes, and promotes the establishment of new symbiotic relationships. At the same time, the higher diversity of the bacterial community in the Pocillopora damicornis makes the microbial community structure change have more bacterial group selection to buffer disease stress and maintain the stability of the microbial community. Therefore, this characteristic of the symbiotic bacterial community of the Pocillopora damicornis can potentially help the Pocillopora damicornis to buffer the disease stress mediated by V. corallivorus and improve its disease tolerance through coral microbial transplantation.

[0024] The relative abundance of Ruegeria sp. Ruegeria will increase after the Pocillopora damicornis is stressed by V. corallivorus, and Ruegeria sp. has been proved to be a coral probiotic that can produce antibacterial active substances and effectively inhibit the proliferation of coral pathogenic bacteria including V. corallivorus. Therefore, in the process of coral microbial transplantation, Ruegeria sp. is potentially involved in the process of coral resisting pathogens and helps to improve the disease tolerance of the Pocillopora damicornis.

[0025] Preferably, during the culture process of step S1, the artificial hatched Caridina nilotica is fed twice a week, and the coral culture time is 15-22 days.

[0026] ​Preferably, in step S3, the physiological state of the corals under Vibrio coralliiyctus stress is evaluated by monitoring and comparing the indicators of the cultured Pocillopora damicornis and Montipora sp., and when significant differences are observed in the indicators of the two corals, corals resistant and susceptible to Vibrio coralliiyctus are screened; the indicators are as follows:

[0027] Maximum quantum yield, zooxanthellae density, coral color index, total superoxide dismutase activity, catalase enzyme activity.

[0028] Preferably, in step S6, the volume of sterile seawater used for resuspension is 2 mL per 1 cm of the surface area S of the coral block. 2 .

[0029] Preferably, in step S7, the method for determining whether the mixed solution V3 can improve the resistance of the susceptible coral is as follows:

[0030] (1) If the state of the coral samples in the blank group and the experimental group is better than that in the control group, it indicates that the potential probiotics in the mixed solution V3 are effective in preventing and treating coral diseases caused by the target pathogenic bacteria.

[0031] (2) If one of the coral samples in the experimental group and the control group is similar to the blank group and fails to cause coral disease, it indicates that the substance in the mixed solution V3 does not have an absolute pathogenic effect on the coral and can be used as a potential probiotic for disease prevention and treatment experiments.

[0032] (3) If the state of the coral samples in the blank group and the experimental group is better than that in the control group, it indicates that the potential probiotics in the mixed solution V3 are effective in preventing and treating coral diseases caused by the target pathogenic bacteria.

[0033] Compared with the prior art, the present application has the following advantages:

[0034] 1. The present application is simple, safe and reliable, has high coral mixed solution extraction efficiency and microbial activity, and has a wide application prospect.

[0035] 2. The screening process of the present application is simple and reliable, and can be tested according to the changes in the phenotypic characteristics of different corals to avoid unsatisfactory screening results.

[0036] 3. The screened Vibrio coralliiyctus susceptible coral has a rapid response efficiency to Vibrio coralliiyctus stress, and can effectively evaluate the effect of the Vibrio coralliiyctus resistant coral mixed solution on improving the resistance of the susceptible coral to Vibrio coralliiyctus. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 Different phenotypic characteristics of D. antillarum and D. claviformis under the same Vibrio corallilyticus stress.

[0038] Figure 2 Changes of zooxanthellae of D. antillarum and D. claviformis under the same Vibrio corallilyticus stress.

[0039] Figure 3 Different phenotypic characteristics of D. antillarum in different treatment groups.

[0040] Figure 4 Maximum quantum yield of D. antillarum in different treatment groups.

[0041] Figure 5 Changes of symbiotic zooxanthellae density of D. antillarum in different treatment groups.

[0042] Figure 6 Different distribution of total superoxide dismutase (T-SOD) activity of D. antillarum in different treatment groups.

[0043] Figure 7 Different distribution of catalase (CAT) activity of D. antillarum in different treatment groups.

[0044] Figure 8 Different distribution of lipid peroxidation (LPO) activity of D. antillarum in different treatment groups. EMBODIMENT

[0045] The application will be further described below in conjunction with the accompanying drawings, and in the examples, the technical means used are all conventional technical means in the art unless otherwise specified. EMBODIMENT

[0046] The embodiment is to improve the Vibrio corallilyticus tolerance of D. antillarum, which is a Vibrio corallilyticus susceptible coral, and specifically includes the following steps:

[0047] (1) Select D. claviformis and D. antillarum in good growth state, and set the water quality conditions of the culture pond as follows:

[0048] Temperature: 26℃, KH: 7.0, pH: 8.2, Ca 2+ : 400ppm, Mg 2+ : 1400ppm, PO4 3- <0.03ppm, NH 3+ <0.15ppm, NO 2- <0.1ppm, NO 3-≈0ppm, light 250W Metal halogen lamp + 4 T5HO, ODYSSEA;

[0049] Set the length of the light: no light = 12h:12h to simulate the natural environment, the collected coral samples in the environment to adapt to 20d, twice a week feeding artificial hatching of the rich year shrimp;

[0050] (2) the solution of coral Vibrio is rejuvenated, and the bacterial liquid after 24h culture is transferred to a 1ml centrifuge tube, centrifuged at 8000rpm for 6min, the supernatant is removed, 330μL of cylinder seawater is added to resuspend the precipitate, and the solution of coral Vibrio is prepared for inoculation;

[0051] (3) the solution of coral Vibrio is inoculated into the horn cup-shaped coral and horn nest coral obtained by step S1 culture, respectively, the horn cup-shaped coral is cultured for 11h, and the horn nest coral is cultured for 24h, and the coral with tolerance to the solution of coral Vibrio and the susceptible coral are selected; by changing the maximum quantum yield, the coral color index, the total superoxide dismutase activity, the hydrogen peroxidase activity and other indexes, the coral with tolerance to the solution of coral Vibrio, the horn nest coral, and the susceptible coral to the solution of coral Vibrio, the horn cup-shaped coral, are screened out.

[0052] (4) the growth state of the selected solution of coral Vibrio resistant coral species, horn nest coral, is good, which is used as a mixed liquid donor for the preparation of mixed liquid;

[0053] The mixed liquid V1(ml) is obtained by washing the coral block with sterile seawater by using a dental cleaner, and then the surface area S(cm 2 ) of the coral block is measured with tin paper. V1 is filtered with neutral filter paper to remove large particles and coral mucus to obtain filtered mixed liquid V2(ml), and then V2 is transferred to a 50ml centrifuge tube, centrifuged at 8000g for 5min, the supernatant is removed, and a certain volume of sterile seawater is resuspended to obtain mixed liquid V3(ml); 1ml of mixed liquid V3 is added to sterile seawater and diluted 10 times to obtain mixed liquid diluent V4, i.e. the solution of coral Vibrio resistant coral mixed liquid.

[0054] The ratio of the volume of sterile seawater used for resuspension to the surface area S of the donor coral is 2mL:1cm 2 .

[0055] (5) Effect evaluation of the improvement of the V. corallivorus resistance of the Pocillopora damicornis after inoculating the V. corallivorus resistant Pocillopora damicornis: the V. corallivorus resistance effect of the V. corallivorus resistant Pocillopora damicornis mixed liquid after inoculation is evaluated by the maximum quantum yield, coral color index, and total superoxide dismutase (T-SOD), catalase (CAT), and lipid peroxide (LPO) activities of the Pocillopora damicornis. If there is a significant difference between the above indexes of the V. corallivorus resistant Pocillopora damicornis mixed liquid group and the non-inoculated V. corallivorus resistant Pocillopora damicornis mixed liquid group, it indicates that the V. corallivorus resistant Pocillopora damicornis mixed liquid can effectively improve the V. corallivorus resistance of the V. corallivorus susceptible Pocillopora damicornis, and that the V. corallivorus resistant Pocillopora damicornis mixed liquid can successfully improve the V. corallivorus resistance of the Pocillopora damicornis. The specific steps are as follows:

[0056] The V. corallivorus resistant Pocillopora damicornis mixed liquid obtained in step (4) is inoculated into the Pocillopora damicornis susceptible to V. corallivorus obtained in step (3), and the Pocillopora damicornis is cultured for 24 h. Then the maximum quantum yield, zooxanthellae density, coral color index, total superoxide dismutase (T-SOD) activity, catalase (CAT) activity, and lipid peroxide (LPO) activity of the cultured Pocillopora damicornis are measured. The anti-disease effect is evaluated according to the significant difference in infection symptoms between the Pocillopora damicornis inoculated with the mixed liquid and the Pocillopora damicornis not inoculated with the mixed liquid. When there is a significant difference between the data of the Pocillopora damicornis inoculated with the mixed liquid and the Pocillopora damicornis not inoculated with the mixed liquid, it indicates that the mixed liquid can improve the V. corallivorus resistance of the susceptible Pocillopora damicornis. Example

[0057] The method of this example is the same as that of Example 1, except that the parameters are set differently. The specific steps are as follows:

[0058] In step (1), the water quality conditions of the culture tank are set as follows: temperature 26℃, KH: 6.7, pH: 8.1, Ca 2+ : 385ppm, Mg 2+ : 1380ppm, PO4 3- <0.03ppm, NH 3+ <0.15ppm, NO 2- <0.1ppm, NO 3- ≈0ppm, and the light is provided by a 250W Metalhalogen lamp + 4 T5HO, ODYSSEA.

[0059] In step (3), the V. corallivorus liquid is inoculated into the Pocillopora damicornis and the Montipora capitata obtained in step S1. The Pocillopora damicornis is cultured for 11 h, and the Montipora capitata is cultured for 24 h.

[0060] Step (5): The V. corallivorus-tolerant coral mixture obtained in step (4) is inoculated into the susceptible coral, Pocillopora damicornis, screened in step (3), and the culture time is 24 h. Embodiment

[0061] The method of this embodiment is the same as that of Embodiment 1, except that the parameters are set differently, as follows:

[0062] Step (1): The water quality conditions of the culture tank are set as follows: temperature 26℃, KH: 7.3, pH: 8.3, Ca 2+ : 415ppm, Mg 2+ : 1420ppm, PO4 3- <0.03ppm, NH 3+ <0.15ppm, NO 2- <0.1ppm, NO 3- ≈0ppm, and illumination is 250W Metalhalogen lamp + 4 T5HO, ODYSSEA.

[0063] Step (3): The V. corallivorus bacterial solution is inoculated into the Pocillopora damicornis and the Montipora capitata obtained in step S1, wherein the Pocillopora damicornis is cultured for 11 h and the Montipora capitata is cultured for 24 h.

[0064] Step (5): The V. corallivorus-tolerant coral mixture obtained in step (4) is inoculated into the susceptible coral, Pocillopora damicornis, screened in step (3), and the culture time is 24 h.

[0065] Application Example 1

[0066] The method of Embodiment 1 is followed for coral culture, wherein:

[0067] The Vibrio-stressed group is the Pocillopora damicornis in step (3) inoculated with seawater for stress culture;

[0068] The microbial transplantation group is the Pocillopora damicornis in step (3) inoculated with the V. corallivorus-tolerant coral mixture prepared in step (4) for culture.

[0069] Then, the data testing and comparison are carried out according to the following steps:

[0070] (1) Photographs are taken to record the Pocillopora capitata and Pocillopora damicornis during the entire process for later analysis of their phenotypic changes.

[0071] The phenotypic changes of the two corals after step (2) V. corallivorus inoculation are shown in Table 1. Figure 1

[0072] ​The phenotype change characteristics of the Vibrio-stressed group and the microorganism transplanted group of the cultured Pocillopora damicornis after step (4)-(5) are shown in Figure 3 .

[0073] (2) The maximum quantum yield (Chlorophyll fluorescence) was measured by Diving-PAM underwater fluorometer (WALZ, Germany), and the test results are shown in Figure 4 .

[0074] (3) The coral color index was obtained after processing the photos. Adobe Photoshop 2022 was used to randomly select 20 color points in the coral sample part of each photo, record the RGB value of the position where each color point was located, calculate the gray value of each color point through the gray value formula Gray = R x 0.3 + G x 0.59 + B x 0.11, and calculate the average gray value of the 20 points to obtain the representative gray value of each photo. All representative gray values were established in origin to obtain a standard color card. The color index of the coral in each photo was the color index grade obtained after comparing the average gray value with the standard color card.

[0075] (4) The seawater was used to rinse part of the coral tissue, and the rinse liquid was centrifuged at 4°C and 1500g for 15min, and then the supernatant was stored in a ultra-low temperature refrigerator at -80°C for determination of the activities of total superoxide dismutase (SOD), catalase (CAT), and lipid peroxide (LPO).

[0076] The activities of SOD, CAT, and LPO were determined by Nanjing Jiancheng total superoxide dismutase (T-SOD) test kit (hydroxylamine method), catalase (CAT) determination kit (visible light method), and lipid peroxide (LPO) determination kit (visible light method). 50ml of filtrate was centrifuged at 4000g and 4°C for 10min, and repeated mirror inspection was performed until the zooxanthellae were clean, and then 4% formaldehyde was used for fixation for later counting. The test results are shown in Figures 6-8 .

[0077] The comprehensive evaluation results are as follows:

[0078] 1、 Figure 1The results show that the PDC and PDE have different phenotypic characteristics under Vibrio challenge. After Vibrio challenge, the PDC shows obvious tentacle retraction, color fading and tissue shedding, while the FC does not show color whitening and tissue shedding, indicating that the PDC has higher susceptibility to Vibrio shanghaii, while the FC has higher tolerance to Vibrio shanghaii.

[0079] 2、 Figure 2 The results show that the symbiotic zooxanthellae density of the PDC and PDE decreases under Vibrio challenge, but the symbiotic zooxanthellae density of the FC is higher than that of the PDC before and after Vibrio challenge.

[0080] 3、 Figure 3 The results show that the PDC inoculated with the FC mixture and the PDC not inoculated with the FC mixture have different phenotypic characteristics under Vibrio challenge. The tentacles of the PDC inoculated with the FC mixture do not retract, and no tissue shedding is observed. The tentacles of the PDC not inoculated with the FC mixture retract, and the tissue is severely shed. This indicates that the mixture of the FC with tolerance to Vibrio shanghaii can effectively improve the tolerance of the PDC susceptible to Vibrio shanghaii to Vibrio shanghaii.

[0081] 4、 Figure 4 The results show that the PDC inoculated with the FC mixture (BV) and the PDC not inoculated with the FC mixture (SV) have different maximum quantum yield changes under Vibrio challenge. The maximum quantum yield of the PDC inoculated with the FC mixture does not decrease significantly, while the maximum quantum yield of the PDC not inoculated with the FC mixture decreases significantly during the experiment. This indicates that the mixture of the FC with tolerance to Vibrio shanghaii can effectively maintain the stability of the photosynthetic rate of the PDC susceptible to Vibrio shanghaii.

[0082] 5、 Figure 5 The results show that the PDC inoculated with the FC mixture (BV) and the PDC not inoculated with the FC mixture (SV) have different zooxanthellae density changes under Vibrio challenge. The symbiotic zooxanthellae density of the PDC inoculated with the FC mixture is significantly higher than that of the PDC not inoculated with the FC mixture. This indicates that the mixture of the FC with tolerance to Vibrio shanghaii can effectively maintain the symbiotic zooxanthellae density of the PDC susceptible to Vibrio shanghaii, further maintaining the stability of the photosynthetic rate.

[0083] 6、 Figure 6The results show that the T-SOD activity of the D. subglobosa (BV) inoculated with the mixed liquid of the P. angulum and the D. subglobosa (SV) not inoculated with the mixed liquid of the P. angulum changes differently under Vibrio stress. The T-SOD activity of the D. subglobosa inoculated with the mixed liquid of the P. angulum is significantly higher than that of the D. subglobosa not inoculated with the mixed liquid of the P. angulum, which indicates that the mixed liquid inoculated with the P. angulum with Vibrio corallinum tolerance can effectively maintain the normal level of active oxygen in the tissue cells of the D. subglobosa, so as to improve the tolerance of the D. subglobosa to Vibrio corallinum.

[0084] 7、 Figure 7 The results show that the CAT activity of the D. subglobosa (BV) inoculated with the mixed liquid of the P. angulum and the D. subglobosa (SV) not inoculated with the mixed liquid of the P. angulum changes differently under Vibrio stress. The CAT activity of the D. subglobosa inoculated with the mixed liquid of the P. angulum is lower than that of the D. subglobosa not inoculated with the mixed liquid of the P. angulum, but there is no significant difference.

[0085] 8、 Figure 8 The results show that the LPO activity of the D. subglobosa (BV) inoculated with the mixed liquid of the P. angulum and the D. subglobosa (SV) not inoculated with the mixed liquid of the P. angulum changes differently under Vibrio stress. The LPO activity of the D. subglobosa inoculated with the mixed liquid of the P. angulum is significantly higher than that of the D. subglobosa not inoculated with the mixed liquid of the P. angulum, which indicates that the mixed liquid inoculated with the P. angulum with Vibrio corallinum tolerance can effectively maintain the LPO activity of the D. subglobosa, and help to maintain the health state of the coral.

[0086] The above examples are only exemplary embodiments of the present application, and are not used to limit the present application, and the protection scope of the present application is defined by the claims. Those skilled in the art can make various modifications or equivalent replacements to the present application within the spirit and protection scope of the present application, and such modifications or equivalent replacements are also regarded as falling within the protection scope of the present application.

Claims

1. A method of increasing the tolerance of corals to Vibrio coralliiyunzae, characterized in that, Comprise the following steps: S1 : Select healthy and well-grown corals of Acropora and Montipora, and culture them under the following growth conditions: temperature: 24-28°C, pH: 8.1-8.3, KH: 7.0±0.3, pH: 8.2±0.1, Ca 2+ : 400±15 ppm, Mg 2+ : 1400±20 ppm, PO4 3- <0.03 ppm, NH 3+ <0.15 ppm, NO 2- <0.1 ppm, NO 3- ≈0 ppm, and use halogen lamps to simulate natural light, with a light duration: dark duration of 12h: 12h. S2: the Vibrio corallii is rejuvenated, and the Vibrio corallii liquid after 24h culture is centrifuged at 8000rpm for 6min, the supernatant is removed, then the precipitate is resuspended in seawater to prepare Vibrio corallii liquid; S3: the Vibrio corallii liquid is inoculated into the Echinomuricea and the Pocillopora corals cultured in step S1 respectively, and cultured for 11-24h, and the corals with resistance and susceptibility to Vibrio corallii are screened out; the indicators of the cultured Echinomuricea and Pocillopora corals are monitored and compared to evaluate the physiological state changes of the corals under Vibrio corallii stress, when the indicators of the two kinds of corals appear significant differences, the corals with resistance and susceptibility to Vibrio corallii can be screened out; The indicators are as follows: maximum quantum yield, zooxanthellae density, coral color index, total superoxide dismutase activity, catalase enzyme activity; S4: the Pocillopora coral with resistance to Vibrio corallii screened out in step S3 is taken as a donor, the coral block is washed with sterile seawater by a dental cleaner to obtain a mixed liquid V1, and then the surface area S of the coral block is measured with tin paper; S5: the mixed liquid V1 is filtered with neutral filter paper to remove large particle impurities and coral mucus to obtain a mixed liquid V2; S6: the mixed liquid V2 is centrifuged at 8000g for 5min, the supernatant is removed, and then resuspended with sterile seawater and shaken to obtain a mixed liquid V3, i.e. a donor mixed liquid; the volume V3 of the donor mixed liquid is 2 times the surface area S of the coral block; S7: the mixed liquid V3 is inoculated into the Echinomuricea coral with susceptibility to Vibrio corallii screened out in step S3 as a test group, the same amount of seawater as the mixed liquid V3 is inoculated into the Echinomuricea coral with susceptibility to Vibrio corallii screened out in step S3 as a blank group, and the mixed liquid V3 and the same amount of Vibrio corallii liquid are mixed and inoculated into the Echinomuricea coral with susceptibility to Vibrio corallii screened out in step S3 as a control group; the corals of the test group, the blank group and the control group are cultured for 24-116h; S8: the maximum quantum yield, the zooxanthellae density, the coral color index, the total superoxide dismutase activity and the catalase enzyme activity of the cultured Echinomuricea corals are measured, and the anti-disease effect is evaluated by analyzing the significant difference in infection symptoms between the corals inoculated with and not inoculated with the mixed liquid V3; when the growth data of the corals inoculated with and not inoculated with the mixed liquid V3 appear significant difference, it indicates that the mixed liquid V3 can improve the resistance of the susceptible corals to Vibrio corallii.

2. The method of increasing the tolerance of corals to V. shiloi according to claim 1, characterized in that: During the culture process of step S1, the artificial hatched Caridina nilotica is fed twice a week, and the coral culture time is 15-22d.

3. The method for increasing the tolerance of corals to V. shiloi according to claim 1, characterized in that: The way to determine whether the mixed liquid V3 can improve the resistance of the susceptible corals is as follows in step S7: (1) if the states of the coral samples of the blank group and the test group are better than those of the control group, it indicates that the potential probiotics in the mixed liquid V3 are effective in preventing and treating the coral diseases caused by the target pathogenic bacteria; (2) If one of the coral samples in the experimental group and the control group is similar to the blank group, and neither can cause coral disease, it means that the substances in the mixed solution V3 do not have an absolute pathogenic effect on coral, and can be used as potential probiotics for coral disease prevention and treatment experiments; (3) If the coral samples in the blank group and the experimental group are better than the control group, it means that the potential probiotics in the mixed solution V3 are effective in preventing and treating coral diseases caused by target pathogenic bacteria.

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