A compound microbial agent for efficiently promoting coral larva attachment metamorphosis and larva growth and application thereof

Abstract

The application discloses a kind of high-efficiency promoting coral larvae attachment metamorphosis and the growth of complex microbial inoculant and application thereof.The bacterium agent comprises Phytobacter sp.SCSIO43973 and Salipiger sp.SCSIO43974 two strains, and preservation numbers are respectively: GDMCC NO:65583 and 65650.The two strains are isolated from healthy antler cup-shaped coral.It is shown that the addition of these strains can significantly improve the attachment rate, metamorphosis rate of planulae, and the survival rate and budding rate of coral larvae.These characteristics show that the bacterium agent has the potential to improve the attachment metamorphosis rate of coral larvae and promote the growth in the field coral reef ecosystem, and has important significance for increasing the amount of larvae supplement, promoting the recovery and sustainable development of coral reef ecosystem.The above, the microbial inoculant of the application promotes the attachment metamorphosis of coral larvae and the growth of larvae, and provides a new technical means for the recovery of coral reef ecosystem.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to the application of compound microbial agents in promoting the attachment metamorphosis and larval growth of coral larvae. Background Technology

[0002] Coral reef ecosystems, often called "rainforests of the ocean," play a vital role in global ecosystems. Covering less than 1% of the ocean, they provide habitat for approximately 25% of the world's marine life, maintaining extremely high biodiversity. Coral reefs are not only crucial for fisheries resources but also provide food and economic income for coastal communities. Furthermore, they function as windbreaks and seawalls, protecting coastlines from storm and wave erosion. Coral reefs also participate in the carbon cycle, enhancing their carbon sink function through organic carbon metabolism and inorganic carbon mineralization, playing a vital role in mitigating global climate change. Therefore, protecting coral reefs is essential for maintaining global ecological balance and human well-being.

[0003] However, globally, statistics show that over 10% of coral reefs have died, and approximately 50% are currently being damaged. Due to climate change, the 1998 coral bleaching event alone caused the death of about 8% of the world's corals. Coral bleaching is becoming more frequent and severe, and the world is currently experiencing the fourth global coral bleaching event on record. Since the beginning of 2023, coral bleaching has been recorded in more than 53 countries and regions, including the United States, Australia, and China. According to a recent report by the Intergovernmental Panel on Climate Change (IPCC), even if global warming is successfully limited to well below 1.5°C, we could still face up to 90% coral reef degradation by 2050. Furthermore, increased land-based debris input from human activities, marine pollution, and overexploitation of coastal resources are also significant factors threatening coral reef environments and causing damage to coral reef ecosystems.

[0004] To reverse the decline of coral reef ecosystems, maintain biodiversity, and enhance ecosystem services, ecological restoration of coral reefs is urgently needed. Currently, coral reef ecosystem restoration methods mainly fall into two categories: natural restoration and artificially assisted restoration. With the emergence of molecular biology techniques such as high-throughput sequencing, microbiome technology has developed rapidly, enabling researchers to gain a deeper understanding of the structure and function of microbial communities. The application of these technologies provides new perspectives and methods for coral reef ecosystem restoration.

[0005] Coral symbiotic organisms include a range of microorganisms such as bacteria, archaea, fungi, protozoa, and viruses. These microorganisms form a long-term mutualistic symbiotic relationship with the coral host and play important roles in the coral reef ecosystem, including key functions such as nutrient cycling and immune regulation. The attachment metamorphosis of coral larvae is extremely important in the coral's growth and development history, as it marks the beginning of the coral's transition from a planktonic to a sessile lifestyle. The attachment metamorphosis and subsequent growth of coral larvae play a fundamental role in the construction and maintenance of coral reefs. This process is crucial for maintaining coral populations and restoring degraded coral reefs, and is key to the health of the coral reef ecosystem. However, the attachment metamorphosis of coral larvae is a complex process influenced by various environmental and biological factors. Existing research has shown that microorganisms can significantly influence the attachment metamorphosis of coral larvae by providing attachment signals, forming biofilms, and secreting inducing substances, which is of great significance for the restoration and stability of coral reef ecosystems.

[0006] Coral larvae growth and attachment depend on a sufficient supply of nitrogen (N) and phosphorus (P) nutrients, which are essential for the photosynthesis and energy conversion processes of the coral's symbiotic zooxanthellae. Zooxanthellae fix carbon dioxide (CO2) and produce organic matter through photosynthesis, providing energy to the coral host, while the coral provides the zooxanthellae with essential nutrients such as nitrogen and phosphorus. This mutually beneficial symbiotic relationship ensures that coral larvae receive sufficient energy and nutrients for growth, which is crucial for their survival and development. Therefore, an adequate supply of nitrogen and phosphorus is a key factor for the healthy growth and effective attachment of coral larvae; insufficient nutrition may limit the developmental potential, attachment success rate, and post-attachment growth of coral larvae. Studies have reported that coral microbiome engineering can enhance the tolerance and resilience of corals under dynamic environmental conditions through precise and controllable manipulation of the microbiome, making it an effective means of protecting and restoring coral reef ecosystems. Therefore, this patent is based on the principle of coral microbiome technology and aims to obtain probiotics (such as nitrogen-fixing and phosphorus-solubilizing bacteria) from coral reef ecosystems to help coral larvae attach and metamorphose and grow, thereby providing strong technical support for the restoration of coral reef ecosystems. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing a compound microbial agent that efficiently promotes the attachment metamorphosis and larval growth of coral larvae and its application. The microbial strain is isolated from healthy staghorn cup coral (Pocilloporadamicornis), which can effectively promote the attachment metamorphosis and larval growth of coral larvae.

[0008] Phytobacter sp.SCSIO43973 was deposited on December 6, 2024, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC 65583), located at No. 100 Xianlie Middle Road, Guangzhou, 510070, China, with accession number GDMCC NO: 65583.

[0009] Salipiger sp.SCSIO43974 was deposited on December 16, 2024 at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC 65583), located at No. 100 Xianlie Middle Road, Guangzhou, 510070, with accession number GDMCC NO: 65650.

[0010] The purpose of this invention is to provide a compound microbial agent that effectively promotes the attachment metamorphosis and larval growth of coral larvae, including Phytobacter sp.SCSIO43973, Salipiger sp.SCSIO43974 or combinations thereof.

[0011] Preferably, the coral larvae and juveniles include, but are not limited to, staghorn cup coral larvae and juveniles.

[0012] Preferably, the bacterial agent is a microbial agent composed of a bacterial solution containing Phytobacter sp. SCSIO43973 and a bacterial solution containing Salipigers sp. SCSIO43974 in a volume ratio of 1:1.

[0013] The present invention also provides the application of Phytobacter sp. SCSIO43973, Salipiger sp. SCSIO43974 or combinations thereof in improving the attachment metamorphosis rate of coral larvae and promoting the growth of coral larvae.

[0014] The present invention also provides a method for improving the attachment metamorphosis rate of coral larvae and promoting the growth of coral larvae, which involves releasing Phytobacter sp. SCSIO43973, Salipiger sp. SCSIO43974, or combinations thereof into the coral larvae culture system.

[0015] Preferably, the coral larvae and coral juveniles include, but are not limited to, staghorn cup coral larvae and juveniles.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] Both SCSIO43973 and SCSIO43974 of the present invention are derived from coral reef ecosystems and isolated from healthy staghorn cup corals. These microbial agents can effectively improve the attachment rate, metamorphosis rate, survival rate, and budding reproduction rate of coral larvae. Therefore, they have great potential to be applied in wild coral reef ecosystems to improve the attachment and metamorphosis rate of coral larvae and promote the growth of coral larvae, thereby promoting the restoration and sustainable development of coral reef ecosystems.

[0018] Preservation Instructions

[0019] Phytobacter sp.SCSIO43973 was deposited on December 6, 2024, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC NO: 65583), located at No. 100 Xianlie Middle Road, Guangzhou, 510070, China.

[0020] Salipiger sp.SCSIO43974 was deposited on December 16, 2024, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC NO: 65650), located at No. 100 Xianlie Middle Road, Guangzhou, 510070, Guangdong Province, China. Attached Figure Description

[0021] Figure 1 The effect of bacterial agent addition on the 24-hour attachment rate of coral planktonic larvae (n=3)

[0022] Figure 2 (A) shows the effect of microbial agent addition on the metamorphosis rate of planktonic larvae (n=3), and (B) shows the metamorphic morphology of planktonic larvae in different experimental groups.

[0023] Figure 3 (A) is Figure 3 The effects of microbial agent addition on the survival of coral larvae and (B) the effects of microbial agent addition on the budding and reproduction of coral larvae. Detailed Implementation

[0024] The following is a further detailed description of the present invention in conjunction with specific preferred embodiments, but it should not be considered that the specific embodiments of the present invention are limited to this.

[0025] Example 1: Isolation and Identification of Coral Symbiotic Microbial Strains

[0026] Healthy staghorn corals (Pocillopora damicornis) were collected from the coral reef ecosystem. After collection, the corals were immediately placed in seawater for temporary holding and brought back to the laboratory as soon as possible. They were then immediately rinsed three times with sterile seawater to remove impurities and other attached organisms from the coral surface. A small piece of coral (approximately 5-10g) was taken with sterile tweezers and wrapped in a sterile zinc sheet. It was then mashed into a paste with a hammer and transferred entirely into a 50ml sterile centrifuge tube. 10ml of sterile seawater was added, and the mixture was shaken to mix thoroughly. This mixture served as the stock solution for isolating symbiotic bacteria.

[0027] Take approximately 1 ml of the sample stock solution and perform six dilutions (10⁻⁶). -1 10 -2 10 -3 10 -4 10 -5 and 10 -6 For each gradient, 200 μL of bacterial suspension was spread onto 2216E medium (see below for specific medium composition) and incubated at 27°C. After obvious colonies appeared on the medium, different single colonies were picked and inoculated onto new medium, and then isolated and purified by streak plating and subculture.

[0028] The obtained pure bacteria can be cultured in LB medium. After collecting a certain amount of bacterial cells through liquid culture, genomic DNA of the strain is extracted using a bacterial DNA extraction kit (Omega Biotek, USA). Amplification is performed using universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGCTACCTTGTTACGACTT-3′) for the 16S rRNA gene. The reaction system is: rTaq Premix 12.5 μL, 27F 0.2 μmol·L⁻¹ -1 1492R 0.2μmol·L -1 1 μL of template DNA was added, and the reaction volume was brought to 25 μL with ddH2O. The PCR reaction conditions were: 94℃ for 5 min, 94℃ for 30 s denaturation, 56℃ for 30 s annealing, 72℃ for 90 s extension, repeated for 30 cycles, with a final extension at 72℃ for 10 min. After successful amplification, the sample was sent to a sequencing company for sequencing. The sequencing results were compared with the EzBioCloud database (https: / / www.ezbiocloud.net / identify) to preliminarily clarify the genetic information of the strain.

[0029] LB medium: 10g tryptone; 5g yeast extract; 25g NaCl; 18g agar; 1L pure water.

[0030] 2216E medium: 5g tryptone; 1g yeast extract; 0.2g FePO4·2H2O; 18g agar; 1L aged seawater; pH 7.2-7.8.

[0031] Based on database comparison analysis and literature review, two bacteria with potential probiotic functions, SCSIO43973 and SCSIO43974, were selected for the addition experiment. The 16S rRNA sequence of SCSIO43973 is shown in SEQ ID NO.1, and the most similar sequence is Phytobacter palmae S29. (T) (KX893413), with a similarity of 97.71%, is a suspected new species and a potential phosphate-solubilizing bacterium; the 16S rRNA sequence of SCSIO43974 is shown in SEQ ID NO.2, and the most similar sequence is Salipiger mangrovisoli 6D45A. (T) The similarity was 99.25%, indicating that they are potential nitrogen-fixing bacteria. Based on the antagonistic experiment results, there was no antagonistic effect between the two strains. Therefore, the two strains were combined to create a microbial agent that can improve the attachment metamorphosis rate of coral larvae and promote their larval growth.

[0032] SCSIO43973, named Phytobacter sp. SCSIO43973, was deposited on December 6, 2024, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC 65583), located at No. 100 Xianlie Middle Road, Guangzhou, 510070, with accession number GDMCC NO: 65583.

[0033] SCSIO43974, named Salipiger sp., was deposited on December 16, 2024, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC 65583), located at No. 100 Xianlie Middle Road, Guangzhou, 510070, with accession number GDMCC NO: 65650.

[0034] Example 2: Effect of bacterial agent on the 24-hour attachment rate of planktonic larvae

[0035] Phytobacter sp. SCSIO43973 and Salipiger sp. SCSIO43974 were inoculated into LB medium and cultured overnight at 27°C and 180 rpm for 12 hours to obtain bacterial suspensions. This yielded single-celled agents of Phytobacter sp. SCSIO43973 and Salipiger sp. SCSIO43974. The Phytobacter sp. SCSIO43973 and Salipiger sp. SCSIO43974 bacterial suspensions were then mixed at a volume ratio of 1:1 to obtain a compound bacterial agent.

[0036] The effects of single and combined microbial agents on the 24-hour attachment rate of planktonic larvae were investigated. Four groups were set up: a control group, a single microbial agent group 1 (SCSIO43973), a single microbial agent group 2 (SCSIO43974), and a combined microbial agent group (a 1:1 combination of SCSIO43973 and SCSIO43974). Each group had three replicates. The experimental setup consisted of 6L coral rearing tanks, with 500 planktonic larvae placed in each tank. 4.5L of seawater was added to the 6L coral rearing tank, and 15 coral substrates were placed in each tank. The same number of planktonic larvae (500 each) were cultured in each tank. Different microbial agents were added to the experimental groups to achieve a final concentration of 10% in the rearing tank. 6 Cells / ml. Aerated culture for 24 hours. After culture, the attached larvae were counted to calculate the 24-hour attachment rate of planktonic larvae. Based on the preliminary effects of the microbial agent, the type of microbial agent for subsequent examples was screened.

[0037] Example 3: Effect of compound microbial agent on metamorphosis rate of planktonic larvae

[0038] Based on the results of Example 2, the effect of the compound microbial agent on the metamorphosis rate of planktonic larvae was investigated. Two groups were set up: a control group and a microbial agent group, with three replicates in each group. The experimental setup consisted of a 6L coral culture tank, with 500 planktonic larvae added to each tank. 4.5L of seawater was added to the 6L coral culture tank, and the culture time was 72 hours. The microbial agent was added at the beginning of the culture to achieve a final concentration of 10% in the culture tank. 6 / ml. After culture, 100 planktonic larvae were taken from each replicate group, and their metamorphosis was observed under a dissecting microscope. The metamorphosis rate and the proportion of tentacles were calculated.

[0039] Example 4: Effects of compound bacterial agent on 15-day survival rate and budding reproduction of coral larvae

[0040] This study investigated the effects of a microbial agent on the 15-day survival rate and budding reproduction of coral larvae. Two groups were established: a control group and a microbial agent group, with three replicates in each group. The experimental setup consisted of a 6L coral culture tank, with 500 planktonic larvae added to each tank. 4.5L of seawater was added to the 6L coral culture tank, and 15 coral substrates were placed in each tank. After the planktonic larvae attached and metamorphosed, 100 metamorphosed larvae from each group were retained for the experiment. A microbial agent was added to the culture system to achieve a final concentration of 10% in the culture tank. 6 The number of coral larvae was counted per ml. Aeration was maintained during cultivation, and the number of larvae was counted every other day. After counting, the water was changed, and a bacterial inoculum was added, maintaining the same concentration throughout the cultivation period. After cultivation, the surviving coral larvae were counted and photographed, and their survival rate and budding reproductive rate were calculated.

[0041] The above analysis yielded the following results:

[0042] The attachment rates of planktonic larvae within 24 hours were 40.95%, 45.70%, 46.94%, and 49.68% for the control group, single-agent group 1, single-agent group 2, and compound-agent group, respectively. There was no significant difference between the control group and single-agent groups 1 and 2. P >0.05), and there was a significant difference between the control group and the compound microbial agent group. P <0.05), such as Figure 1 As shown in the figure, this result indicates that adding a single microbial agent can improve the attachment rate of planktonic larvae to some extent, but without a significant promoting effect; while the addition of a compound microbial agent can significantly improve the attachment rate of planktonic larvae within 24 hours. Compared with the control group, the attachment rate of the compound microbial agent group increased by approximately 9.03%, showing that the compound microbial agent has a significant promoting effect on the attachment of planktonic larvae, thus supporting the effectiveness of the compound microbial agent in improving the attachment rate of planktonic larvae. These findings provide experimental evidence for further research on the application of microbial agents in coral reef ecosystem restoration. Based on this, the microbial agent group in our subsequent examples refers to the compound microbial agent group (a 1:1 volume ratio of SCSIO43973 and SCSIO43974).

[0043] The metamorphosis rates of planktonic larvae in the control group and the fungal agent group after 72 hours were 11.60% and 35.55%, respectively, and the proportions of extended tentacles were 3.32% and 16.28%, respectively. Figure 2 As shown in (A), based on statistical analysis, the metamorphosis rate of larvae in the fungal agent group was significantly different from that in the control group (P<0.01), and the ratio of extended tentacles was significantly different from that in the control group (P<0.05). Figure 2 As shown in (B), based on dissecting microscopic observation, it was found that the metamorphosis of larvae in the fungal agent group was earlier than that in the control group. The results indicate that the addition of fungal agents can effectively increase the metamorphosis rate and tentacles ratio of planktonic larvae, and shorten their growth cycle to a certain extent.

[0044] like Figure 3 As shown in (A), the survival rates of coral larvae on day 3 in the control group and the fungicide group were 84.95% and 94.20%, respectively, and their survival rates on day 15 were 100.97% and 132.61%, respectively (due to budding). Because the coral larvae begin budding reproduction after attachment metamorphosis, the survival rate on day 15 was higher in the fungicide group than in the control group; simultaneously, as... Figure 3 As shown in (B), the budding rate of coral larvae was higher in the fungicide group than in the control group. The results indicate that the addition of the fungicide can improve the survival rate of coral larvae and promote their budding reproduction.

[0045] This invention comprehensively analyzes the effects of combined microbial agents on the 24-hour attachment rate, metamorphosis rate, survival, and budding reproduction of coral planktonic larvae. The results show that the compound microbial agent prepared from the combination of *Phytobacter sp. SCSIO43973* and *Salipiger sp. SCSIO43974* strains isolated from coral reef ecosystems significantly improves the 24-hour attachment rate, 72-hour metamorphosis rate, and tentacle ratio of planktonic larvae, while also promoting larval survival and budding reproduction. These effects contribute to increasing the replenishment rate of new coral individuals and promoting coral reef ecosystem restoration. The compound microbial agent provided by this invention, as an effective microbial resource, has significant scientific value and practical application implications for marine ecological protection and coral reef restoration. By utilizing these specific microbial strains, the attachment and metamorphosis processes of coral larvae can be enhanced, increasing their survival rate in the natural environment, which is crucial for maintaining and restoring the biodiversity and ecological functions of coral reefs.

[0046] 16S rRNA

[0047] Phytobacter sp.SCSIO43973

[0048]

[0049] >Salipiger sp.SCSIO43974

[0050]

Claims

1. A highly efficient compound microbial agent for promoting attachment metamorphosis and larval growth in coral larvae, characterized in that, include Phytobacter sp. SCSIO43973 and Salipiger sp. SCSIO43974, as described Phytobacter sp.SCSIO43973, accession number: GDMCC NO: 65583, the aforementioned Salipiger sp. SCSIO43974, accession number: GDMCC NO: 65650.

2. The compound microbial agent according to claim 1, characterized in that, The coral larvae and juveniles mentioned include, but are not limited to, staghorn cup corals ( Pocillopora damicornis Larvae and young.

3. The compound microbial agent according to claim 1, characterized in that, The aforementioned Phytobacter bacterial culture of sp.SCSIO43973 and Salipiger The bacterial culture of sp. SCSIO43974 was mixed at a volume ratio of 1:

1.

4. The application of any of the composite microbial agents described in claims 1-3 in improving the attachment and metamorphosis rate of coral larvae and promoting the growth of coral larvae, wherein the corals include, but are not limited to, staghorn cup coral larvae and juveniles.

5. A method for increasing the attachment metamorphosis rate of coral larvae and promoting the growth of coral larvae, characterized in that, The method involves releasing any of the composite microbial agents described in claims 1-3 into a coral larval culture system, wherein the corals include, but are not limited to, staghorn cup coral larvae and juveniles.

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