Application of endophytic bacteria in assisting corals to resist heat stress

Abstract

The application discloses application of animal endosymbiont in assisting corals to resist heat stress. The application significantly improves the resistance of corals to high-temperature environment by applying the animal endosymbiont SCSIO 12664, and helps to protect the corals from the influence of heat stress. The application introduces the application of the animal endosymbiont SCSIO 12664, and the technical scheme aims to overcome the defects of the prior art, improve the resistance of corals to high-temperature heat stress, reduce the negative influence on the ecosystem, and achieve the goal of more sustainable and effective protection of corals.

Description

Technical Field

[0001] This invention relates to the field of marine probiotic technology, specifically the application of the animal endomonad SCSIO 12664 for coral heat resistance. The method of this invention can be widely applied in marine ecosystem management and coral conservation, providing an innovative solution to combat the threats posed by global climate change. Background technology:

[0002] Coral reefs play a vital role in providing habitats for a wide variety of marine species and protecting coastlines from storm damage. In recent decades, coral reef ecosystems have experienced significant deterioration globally, primarily due to the effects of ocean warming caused by global climate change. Rising temperatures disrupt the symbiotic relationship between corals and their endosymbiotic photosynthetic algae, leading to coral bleaching. These endosymbiotic algae provide photosynthetic products that meet over 90% of the host's nutritional needs. Rising temperatures cause prolonged bleaching, ultimately leading to coral death. Given the ecological and economic importance of coral reef ecosystems, measures must be taken to protect them and mitigate future decline in the face of global climate change.

[0003] To effectively address this issue, we are committed to finding innovative bio-assisted technologies to enhance corals' resistance to heat stress. The "coral probiotic hypothesis" suggests that the coral host's adaptability to environmental disturbances can be enhanced by modulating the coral microbiome. Based on this view, manipulating the coral microbiome is considered a promising approach to improving the stress resistance and resilience of the entire coral organism. In previous studies, we isolated and cultured a strain of *Endozoicomonadaceae*. *Endozoicomonadaceae* are common symbionts in corals and are considered an indicator of coral health. They typically thrive in healthy coral tissues but decrease in stressed, diseased, or bleached corals. *Endozoicomonadaceae* may provide its coral host with amino acids and B vitamins, thus offering protection against pathogens. Furthermore, they may participate in the coral sulfur cycle by metabolizing dimethyl thiopropionate (DMSP) into dimethyl sulfide (DMS). Based on their identified functions, *Endozoicomonadaceae* holds promise as a powerful tool to help corals overcome the negative effects of high-temperature environments.

[0004] The concept behind this invention is based on in-depth research into the interaction between animal endomonads and corals, and a profound understanding of heat stress response mechanisms. By leveraging the potential of these animal endomonads, we hope to provide an innovative and sustainable approach to protecting coral ecosystems, enabling them to better adapt to changing climate conditions. The successful application of this technology is expected to be an important measure for protecting corals and marine ecosystems, making a positive contribution to maintaining global marine biodiversity and ecological balance. Summary of the Invention:

[0005] This invention relates to the application of an animal endomonad, SCSIO 12664, which has been successfully isolated and cultured. This animal endomonad has been found to be able to live in symbiosis with corals and exhibits excellent resistance to heat stress under high-temperature conditions. Introducing this animal endomonad into coral ecosystems can effectively improve coral adaptability to high temperatures and mitigate heat stress-induced damage.

[0006] To achieve the above-mentioned objectives of the present invention, the present invention adopts the following technical solution:

[0007] This invention provides the application of Endozoicomonadaceae Marinivarius SCSIO12664 in assisting corals to withstand high-temperature environments.

[0008] Preferably, the endocellular SCSIO 12664 bacterial solution is introduced into the coral habitat to establish a symbiotic relationship with the coral and help the coral resist high-temperature environments.

[0009] This invention also provides the application of Endozoicomonadaceae montiporae SCSIO12664 in the preparation of formulations that help corals resist high-temperature environments.

[0010] Preferably, the coral is a staghorn cup-shaped coral.

[0011] Preferably, the animal endomonad SCSIO 12664 is an animal endomonad SCSIO 12664 bacterial suspension.

[0012] Preferably, the method for preparing the animal endomonad SCSIO 12664 bacterial suspension is as follows:

[0013] Animal endomonads SCSIO 12664 were inoculated into MA medium and cultured to obtain animal endomonads SCSIO 12664 bacterial suspension.

[0014] A further preferred method involves culturing and enriching animal endomonads SCSIO12664 in MA medium at 25°C, with a bacterial concentration of 10. 7 Cells / mL

[0015] The beneficial effects of this invention are as follows:

[0016] 1. Improve coral heat resistance: This invention significantly improves the coral's resistance to high-temperature environments by applying animal endomonads SCSIO 12664, which helps protect corals from the effects of heat stress.

[0017] 2. Sustainability: Strain SCSIO 12664 has been successfully isolated and cultured in previous studies, demonstrating its sustainability and stability, and providing long-term ecological protection for coral ecosystems.

[0018] 3. Simplified operation: This invention eliminates the need for complex strain screening and cultivation processes, as the strains used are already in pure culture, simplifying the implementation steps and reducing operational difficulty.

[0019] 4. Eco-friendly: The use of animal endomonads SCSIO 12664 is an eco-friendly approach that helps maintain and promote the health of coral ecosystems.

[0020] 5. Wide range of applications: This technical solution is not only applicable to specific types of corals, but can also be extended to other coral species to improve their adaptability to heat stress.

[0021] The animal endomonad of this invention, Endozoicomonadaceae Marinivarius SCSIO 12664, is disclosed in the literature *Cultured Bacteria Provide Insight into the Functional Potential of the Coral-Associated Microbiome*, Jie Li, Yiyang Zou, Jian Yang, Qiqi Li, David G. Bourne, Michael Sweet Cong Liu, Anjie Guo, Si Zhang, July / August 2022, Volume 7, Issue 4, Functional Potential of Coral-Associated Bacteria. The applicant also holds and warrants that it will be made available to the public for 20 years from the date of application. Attached Figure Description

[0022] Figure 1 These are results of confocal laser scanning microscopy observation of coral fragments. (A, C) Coral fragments treated with SCSIO 12664, collected on day 24 at 32°C; (B, D) Coral fragments without introduced animal endomonads, collected on day 24 at 32°C; TAMRA / SE signal (yellow), coral GFP (green), algal chlorophyll (red). White arrows indicate TAMRA / SE-tagged cells.

[0023] Figure 2These are physiological and ecological indicators of corals. (A) Photograph of a coral fragment; (B) Color of the coral fragment according to the coral color chart; (C) Symbiotic algae density, ns indicates no significant difference; (D) Photosynthetic efficiency of the coral fragment. Detailed Implementation

[0024] Current technologies are significantly inadequate in addressing the high-temperature stress caused by global climate change. Many coral species have low tolerance to high temperatures, leading to an increased risk of bleaching and death, which directly threatens the stability of the entire coral ecosystem. Existing technologies typically require complex and expensive treatment processes, including human intervention on the corals, the use of chemicals, or other time-consuming and labor-intensive methods. These processes are not only difficult to implement but may also pose environmental risks when applied on a large scale.

[0025] Therefore, by introducing the animal endomonad SCSIO 12664, this invention aims to overcome the shortcomings of the prior art, improve the coral's resistance to high-temperature heat stress, reduce the negative impact on the ecosystem, and achieve a more sustainable and effective goal of coral protection.

[0026] To make the objectives, features, and advantages of the present invention clearer, the specific embodiments of the present invention will be described in more detail below with reference to more specific examples. Many specific details are set forth in the following description to provide a thorough understanding of the present invention; however, the present invention can be practiced in many other ways than those described. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0027] Example 1:

[0028] 1. Introduction Trial

[0029] a. Preparations:

[0030] Animal endomonads were obtained and cultured to ensure their purity and activity. The animal endomonads were labeled with 5(6)-TAMRA-SE fluorescent staining.

[0031] Prepare the staghorn cup coral as a host and ensure it is in good health.

[0032] b. Introduction Experiment Design:

[0033] The corals were divided into experimental and control groups.

[0034] Experimental group: Animal endophytic bacteria SCSIO 12664 were introduced into corals at 32℃.

[0035] Control group: 32℃ without inoculation of animal endomonads SCSIO 12664.

[0036] c. Implementation steps:

[0037] 1. Planting Experiment Procedure

[0038] The collected staghorn cup corals were cut into 50 segments, each 5-6 cm high. These segments were then fixed to clay substrates using coral gel (Alonfa, GEL-10) and evenly distributed in two 60L tanks, with 25 coral segments in each tank. Artificial seawater was added, maintaining a salinity of 31-33‰. The light intensity was adjusted to 150 μmol photons / m². -2 s -1 The flow rate was set at 40 liters per minute. Subsequently, under constant temperature conditions of 25°C, the coral fragment adapted to the new aquatic environment for four days. The water temperature rapidly increased from 25°C to 29°C within one day. Afterward, the water temperature gradually increased from 29°C to 32°C over three days and remained at 32°C for two days.

[0039] On the tenth day, the coral fragments from each tank were aliquoted into five tanks (five coral fragments per tank) as parallel samples. Then, the coral fragments from the experimental group were placed into a 250 mL sterile plastic culture bottle. 1 mL of fluorescently labeled bacterial suspension (containing approximately 10...) was added... 7 (One cell per section) was gently poured into the coral fragment, ensuring complete coverage. Then, approximately 100 ml of sterile seawater was added to completely submerge the coral fragment. The bottle was sealed and returned to the experimental tank. After 45 minutes, the bacterial inoculation process was complete; the coral fragment was removed from the bottle and returned to the tank. Coral fragments in the control group were inoculated with 0.2 μm filtered, autoclaved artificial seawater. All coral fragments were cultured at 32°C for 14 days, until the end of the experiment on day 24.

[0040] Five coral fragments were randomly selected from both the control and experimental groups one day before bacterial inoculation (T1: day 10) and 14 days after inoculation (T2: day 24). These coral fragments were detached from the clay base and gently rinsed with autoclaved artificial seawater. Then, each fragment was cut into three sections, approximately 2 cm in length, using a sterilized bone scalpel. One section was placed on ice, cut into smaller pieces, and then immersed in sterilized artificial seawater for symbiotic algae density determination.

[0041] 2. Detection of symbiotic microbial colonization;

[0042] The coral fragments were carefully washed with sterile artificial seawater filtered through 0.22 μm and autoclaved, and then placed in a chamber cover slide (Thermo Scientific). TMThe images were processed using LAS imaging software (Leica, Germany). The TAMRA-SE target signal (excitation: 551 nm, emission: 578 nm), coral tissue autofluorescence (excitation: 488 nm, emission: 525 nm), and symbiotic algal autofluorescence (excitation: 488 nm, emission: 679 nm) were detected. Coral tissue without TAMRA-SE-labeled bacteria served as a negative control. The experimental results are as follows: Figure 1 As shown, from Figure 1 It can be seen that the animal endomonad SCSIO 12664 successfully colonized coral tissue under high temperature conditions.

[0043] 3. Detection of physiological and health indicators of experimental corals

[0044] Coral color was graded into five levels, from D5 (healthy) to D1 (severely bleached), according to the coral color scale proposed by Siebeck et al. in 2006. Photosynthetic efficiency of corals was measured daily using pulse amplitude modulation (PAM) fluorescence, with 10 replicates randomly tested per group. Symbiotic algae density in coral fragments was determined using a microscopic counting method: coral fragments were thoroughly washed with sterile artificial seawater using a sterile syringe until the skeleton was fully exposed; subsequently, the solution containing tissue fragments was repeatedly aspirated with a syringe for 5 minutes to homogenize the coral tissue; 10 μL aliquots of the homogenized solution were placed in a hemocytometer, and the symbiotic algae cells in each sample were counted five times using five droplets; the surface area of ​​the coral fragments was measured using the aluminum foil coating method, and the number of symbiotic algae cells per unit area was obtained by normalizing the cell count to the surface area of ​​the coral fragments.

[0045] On day 24 (T2), coral fragments exposed to high temperature stress without SCSIO 12664 mainly exhibited D2 grade (59%), while most branches of coral fragments inoculated with SCSIO 12664 were D3 grade. The photosynthetic efficiency of coral fragments inoculated with SCSIO 12664 was significantly higher than that of those without, and the symbiotic algae density of coral fragments inoculated with SCSIO 12664 was also higher on day 24. Figure 2 This indicates that the addition of SCSIO 12664 improves the coral's resistance to high-temperature environments.

Claims

1. A strain of animal endophytic bacteria ( Endozoicomonadaceae Marinivarius The application of SCSIO 12664 in assisting corals to withstand high-temperature environments, wherein the corals are staghorn cup corals.

2. The application according to claim 1, characterized in that, The method involves introducing animal endomonads SCSIO 12664 bacterial solution into coral habitats to establish a symbiotic relationship with corals and help them withstand high-temperature environments.

3. Application of animal endomonads SCSIO 12664 in the preparation of agents to help corals resist high-temperature environments, wherein the coral is a staghorn cup coral.

4. The application according to claim 3, characterized in that, The animal endomonad SCSIO 12664 mentioned above is an animal endomonad SCSIO 12664 bacterial culture.

5. The application according to claim 4, characterized in that, The method for preparing the animal endomonad SCSIO 12664 bacterial suspension is as follows: animal endomonad SCSIO 12664 is inoculated into MA medium and cultured to obtain animal endomonad SCSIO 12664 bacterial suspension.

6. The application according to claim 5, characterized in that, Animal endomonads SCSIO 12664 were cultured and enriched in MA medium at 25°C, with a bacterial concentration of 10. 7 Cells / mL

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