Compound microbial agent capable of efficiently promoting attachment metamorphosis and larva growth of coral larvae and application of compound microbial agent

Abstract

The invention discloses a compound microbial agent for efficiently promoting attachment and metamorphosis of coral larvae and growth of the larvae and application of the compound microbial agent. The microbial agent comprises two strains, namely, Phytobacter sp.SCSIO43973 and Salipiger sp.SCSIO43974, and the preservation numbers of the two strains are respectively as follows: GDMCC (China General Microbiological Culture Collection Center) NO: 65583 and 65650. The two strains are separated from healthy coral deerhorn-shaped corals. Researches show that by adding the strains, the adhesion rate and the metamorphosis rate of planula larvae and the survival rate and the germination reproduction rate of coral larvae can be remarkably increased. The characteristics show that the microbial agent has the potential of improving the attachment metamorphosis rate of coral larvae and promoting growth in a field coral reef ecological system, and has important significance in increasing the larva supplement amount of the damaged coral reef ecological system and promoting the recovery and sustainable development of the coral reef ecological system. In conclusion, the microbial agent provided by the invention provides a new technical means for recovery of a coral reef ecosystem by promoting attachment and metamorphosis of coral larvae and growth of the larvae.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to the application of a composite microbial agent in promoting the attachment metamorphosis and larval growth of coral larvae. Background Art

[0002] Coral reef ecosystems are known as "tropical rainforests in the ocean" and play a vital role in the global ecosystem. They cover less than 1% of the ocean area, but provide habitats for about 25% of the world's marine life and maintain extremely high biodiversity. Coral reefs are not only important for fishery resources, but also provide food and economic income for coastal communities. In addition, they also have the function of windbreaks and embankments, which can protect coastlines from storms and wave erosion. Coral reefs can also participate in the carbon cycle, enhance carbon sink function through organic carbon metabolism and inorganic carbon mineralization, play an important role in the carbon cycle, and help mitigate global climate change. Therefore, protecting coral reefs is crucial to maintaining global ecological balance and human well-being.

[0003] However, currently, according to statistics, more than 10% of coral reefs have died and about 50% of coral reefs are being destroyed worldwide. Affected by climate change, the coral bleaching event in 1998 alone caused the death of about 8% of the world's corals. Coral reef bleaching has become more frequent and severe, and the world is currently experiencing the fourth global coral bleaching event in recorded history. Since the beginning of 2023, coral reef 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 controlled within 1.5℃, we may still face up to 90% of coral reef degradation by 2050. In addition, the increase in terrestrial debris input caused by human activities, seawater pollution and excessive development of coastal resources are also important reasons for threatening the growth environment of coral reefs and causing the destruction of coral reef ecosystems.

[0004] In order to reverse the trend of coral reef ecosystem decline, maintain biodiversity, and enhance the service functions of the ecosystem, it is urgent to carry out ecological restoration of coral reefs. At present, the restoration methods of coral reef ecosystems mainly include natural restoration and artificial assisted restoration. With the emergence of molecular biology technologies such as high-throughput sequencing technology, microbiome technology has developed rapidly, enabling researchers to have a deeper understanding of the structure and function of microbial communities. The application of these technologies provides new perspectives and methods for the restoration of coral reef ecosystems.

[0005] Coral symbionts include a series of microorganisms such as bacteria, archaea, fungi, protozoa and viruses. These microorganisms have formed a long-term mutually beneficial symbiotic relationship with the coral host and play an important role 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 history of coral growth and development, because it is the beginning of the transition from the planktonic stage to the fixed life in the coral life cycle. 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 to the maintenance of coral populations and the restoration of degraded coral reefs, and is the key to the health of coral reef ecosystems. However, the attachment metamorphosis of coral larvae is a complex process that is affected by a variety of environmental and biological factors. Studies have shown that microorganisms can significantly affect the attachment metamorphosis of coral larvae by providing attachment signals, forming biofilms and secreting inducing substances, which is of great significance to the restoration and stability of coral reef ecosystems.

[0006] The growth and attachment of coral larvae depend on an adequate supply of nitrogen (N) and phosphorus (P), which are essential for the photosynthesis and energy conversion process of the coral symbiotic zooxanthellae. Zooxanthellae fix carbon dioxide (CO2) and produce organic matter through photosynthesis, while providing energy to the coral host, while corals provide the zooxanthellae with essential nutrients such as nitrogen and phosphorus. This mutualistic relationship ensures that coral larvae can obtain sufficient energy and nutrients for growth, which is essential for their survival and development. Therefore, an adequate supply of nitrogen and phosphorus nutrients is a key factor in the healthy growth and effective attachment of coral larvae. Insufficient nutrition may limit the developmental potential and attachment success rate of coral larvae and the growth of larvae after attachment. It has been reported that coral microbiome engineering can improve the tolerance and resilience of corals under dynamic environmental conditions by precisely and controllably manipulating the microbiome, which is an effective means to protect and repair 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 phosphate-solubilizing bacteria) derived from coral reef ecosystems by isolating them, to assist the attachment and metamorphosis of coral larvae and the growth of juveniles, thereby providing strong technical support for the restoration of coral reef ecosystems. Summary of the invention

[0007] The object of the present invention is to provide a method for preparing a composite microbial agent for efficiently promoting the attachment metamorphosis and juvenile growth of coral larvae and its application, wherein the microbial strain is isolated from a healthy Acropora scalycopersicum (Pocilloporadamicornis), which can effectively promote the attachment metamorphosis and juvenile growth of coral larvae.

[0008] Phytobacter sp.SCSIO43973 was deposited on December 6, 2024 in Guangdong Microbiological Culture Collection Center (GDMCC 65583), located at No. 100, Xianlie Middle Road, Guangzhou City, Postal Code: 510070, and the deposit number is: GDMCC NO: 65583.

[0009] Salipiger sp.SCSIO43974 was deposited on December 16, 2024 in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC 65583), located at No. 100, Xianlie Middle Road, Guangzhou City, Postal Code: 510070, and the deposit number is: GDMCC NO: 65650.

[0010] The object of the present invention is to provide a composite microbial agent for efficiently promoting the attachment and metamorphosis of coral larvae and the growth of larvae, comprising the above-mentioned Phytobacter sp. SCSIO43973, Salipiger sp. SCSIO43974 or a combination thereof.

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

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

[0013] The present invention also provides the use of the above-mentioned Phytobacter sp. SCSIO43973, Salipiger sp. SCSIO43974 or their combination in improving the attachment and metamorphosis rate of coral larvae and promoting the growth of coral larvae.

[0014] The invention also provides a method for improving the attachment and metamorphosis rate of coral larvae and promoting the growth of coral larvae, which comprises releasing Phytobacter sp. SCSIO43973, Salipiger sp. SCSIO43974 or a combination of these bacterial agents into a culture system of coral larvae.

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

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

[0017] The SCSIO43973 and SCSIO43974 of the present invention are both derived from coral reef ecosystems and separated from healthy Acropora stolonifera. The bacterial agent can effectively improve the attachment rate, metamorphosis rate, survival rate and budding reproduction rate of coral larvae. Therefore, it has great potential for application in wild coral reef ecosystem environments to improve the attachment and metamorphosis rate of coral larvae and promote the growth of coral larvae, thereby promoting the recovery and sustainable development of coral reef ecosystems.

[0018] Collection Instructions

[0019] Phytobacter sp.SCSIO43973 was deposited on December 6, 2024 in Guangdong Provincial Microbiological Culture Collection Center (GDMCC NO: 65583), with the address at No. 100 Xianlie Middle Road, Guangzhou City, Postal Code: 510070, and the deposit number is: GDMCCNO: 65583.

[0020] Salipiger sp.SCSIO43974 was deposited on December 16, 2024 in Guangdong Provincial Microbiological Culture Collection Center (GDMCC NO: 65650), with the address at No. 100 Xianlie Middle Road, Guangzhou City, Postal Code: 510070, and the deposit number is: GDMCC NO: 65650. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Effect of bacterial agent addition on 24h attachment rate of coral planula larvae (n=3)

[0022] Figure 2 (A) Effect of bacterial agent addition on metamorphosis rate of planula larvae (n=3) and (B) Metamorphosis morphology of planula larvae in different experimental groups

[0023] Figure 3 (A) Yes Figure 3 Effects of bacterial addition on the survival of coral larvae and (B) Effects of bacterial addition on the budding and reproduction of coral larvae DETAILED DESCRIPTION

[0024] The following content is a further detailed description of the present invention in combination with specific preferred embodiments, and it cannot be considered that the specific embodiments of the present invention are limited thereto.

[0025] Example 1 Isolation and identification of coral-associated microbial strains

[0026] Healthy staghorn cup corals (Pocillopora damicornis) were collected from the coral reef ecosystem. After the corals were collected and brought ashore, they were immediately placed in seawater for temporary care and brought back to the laboratory as soon as possible. They were immediately rinsed with sterile seawater for three times to remove impurities and other attachments on the surface of the corals. A small piece of coral (about 5-10g) was taken with sterile tweezers and wrapped in a sterile zinc sheet. It was smashed into a paste with a hammer and then all transferred into a 50ml sterile centrifuge tube. 10ml of sterile seawater was added and shaken to mix well as the coral sample mother solution for separating symbiotic bacteria.

[0027] Take about 1 ml of sample stock solution and dilute it in 6 steps (10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 and 10 -6 ). Take 200ul of bacterial solution from each gradient and spread it on 2216E medium (see below for specific medium components), place it in a 27℃ incubator for culture, and after obvious colonies appear on the medium, pick different single colonies and inoculate them on new medium, and separate and purify them by plate streaking method and subculture method.

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

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

[0030] 2216E culture medium: 5 g tryptone; 1 g yeast extract; 0.2 g FePO4·2H2O; 18 g agar; 1 L Chenhai water; PH 7.2-7.8.

[0031] Based on the database comparison analysis results and literature review, two strains of bacteria with potential probiotic functions, SCSIO43973 and SCSIO43974, were selected for addition experiments. The sequence of 16s rRNA 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-soluble bacterium; the sequence of 16s rRNA of SCSIO43974 is shown in SEQ ID NO.2, and the most similar sequence is Salipiger mangrovisoli 6D45A (T) , with a similarity of 99.25%, which is a potential nitrogen-fixing bacteria; based on the results of the antagonism experiment, there is no antagonism between the two, so the two strains were combined to create a microbial agent that can increase the attachment and metamorphosis rate of coral larvae and promote the growth of their larvae.

[0032] SCSIO43973 was named Phytobacter sp. SCSIO43973 and was deposited on December 6, 2024 in the Guangdong Microbiological Culture Collection Center (GDMCC 65583), located at No. 100, Xianlie Middle Road, Guangzhou City, Postal Code: 510070, and the collection number is: GDMCC NO: 65583.

[0033] SCSIO43974 was named Salipiger sp. SCSIO43974 and was deposited on December 16, 2024 in the Guangdong Microbiological Culture Collection Center (GDMCC 65583), located at No. 100, Xianlie Middle Road, Guangzhou City, Postal Code: 510070, and the deposit number is: GDMCC NO: 65650.

[0034] Example 2 Effect of bacterial agents on the 24-hour attachment rate of planula larvae

[0035] Phytobacter sp.SCSIO43973 and Salipiger sp.SCSIO43974 were inoculated into LB medium respectively, and placed in a shaking incubator at 27°C and 180 rpm for overnight culture for 12 hours to obtain bacterial liquid. That is, a single bacterial agent of Phytobacter sp.SCSIO43973 and a single bacterial agent of Salipiger sp.SCSIO43974 were obtained, and the bacterial liquids of Phytobacter sp.SCSIO43973 and Salipiger sp.SCSIO43974 were mixed in a volume ratio of 1:1 to obtain a composite bacterial agent.

[0036] The effects of single and compound bacterial agents on the 24-hour attachment rate of plankton larvae were studied. Four groups were set up in the experiment, namely the control group, single bacterial agent group 1 (SCSIO43973), single bacterial agent group 2 (SCSIO43974) and compound bacterial agent group (a 1:1 combination of SCSIO43973 and SCSIO43974). Three replicates were set up in each group. The experimental device was a 6L coral culture tank, and 500 plankton larvae were placed in each tank. 4.5L of seawater was added to the 6L coral culture tank, and 15 coral bases were placed, and the same number of plankton larvae (500) were cultured respectively; different bacterial agents were added to the experimental groups, so that the final concentration of the bacterial agent added to the culture tank was 10 6 The culture was carried out under aeration for 24 hours. After the culture was completed, the attached larvae were counted to calculate the attachment rate of planula larvae for 24 hours. Based on the preliminary effect of the microbial agent, the types of microbial agents used in the subsequent examples were screened.

[0037] Example 3 Effect of the composite bacterial agent on the metamorphosis rate of planula larvae

[0038] Based on the results of Example 2, the effect of the composite bacterial agent on the metamorphosis rate of planula larvae was studied. The experiment set up two groups, a control group and a bacterial agent group, with three replicates in each group. The experimental device was a 6L coral culture tank, and 500 planula larvae were added to each tank. 4.5L of seawater was added to the 6L coral culture tank. The experimental culture time was 72h. The bacterial agent was added at the beginning of the culture to make the final bacterial concentration in the culture tank 10 6 After the culture was completed, 100 planula larvae were collected from each replicate group, and the metamorphosis of the planula larvae was observed by taking photos under a dissecting microscope, and the metamorphosis rate and the proportion of extended tentacles were calculated.

[0039] Example 4 Effect of the composite bacterial agent on the 15-day survival rate and budding reproduction of coral larvae

[0040] The effect of the microbial agent on the survival rate and budding reproduction of coral larvae at 15 days was studied. There were three replicates in each group, with a control group and a microbial agent group. The experimental device was a 6L coral culture tank, and 500 planula larvae were added to each tank. 4.5L of seawater was added to the 6L coral culture tank, and 15 coral bases were placed. After the planula larvae attached and metamorphosed, 100 larvae attached and metamorphosed were retained in each group for the experiment. The microbial agent was added to the culture system to make the final concentration of the bacteria in the culture tank 10 6 / ml. During the culture period, aeration was performed and the number of coral larvae was counted every other day. After the counting, the water was changed and the bacterial agent was added. The same concentration of the bacterial agent was maintained during the culture period. After the culture period, the surviving coral larvae were counted and photographed, and the survival rate and budding reproduction rate were calculated.

[0041] The above analysis yields the following results:

[0042] The attachment rates of planula larvae in the control group, single bacterial agent group 1, single bacterial agent group 2 and compound bacterial agent group within 24 hours were 40.95%, 45.70%, 46.94% and 49.68%, respectively. There was no significant difference between the control group and single bacterial agent group 1 and single bacterial agent group 2 ( P >0.05), and there was a significant difference between the control group and the compound bacterial agent group ( P <0.05), such as Figure 1 As shown. This result shows that the addition of a single bacterial agent can increase the attachment rate of plankton larvae to a certain extent, but there is no significant promoting effect; while the addition of a composite bacterial agent can significantly increase the attachment rate of plankton larvae within 24 hours. Compared with the control group, the attachment rate of the composite bacterial agent group increased by about 9.03%, showing that the composite microbial agent has a significant promoting effect on the attachment of plankton larvae, thereby supporting the effectiveness of the composite bacterial agent in increasing the attachment rate of plankton larvae. These findings provide an experimental basis for further research on the application of microbial agents in the restoration of coral reef ecosystems. Based on this, the bacterial agent groups in our subsequent examples refer to the composite bacterial agent group (a combination of SCSIO43973 and SCSIO43974 with a volume ratio of 1:1).

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

[0044] like Figure 3 As shown in (A), the survival rates of coral larvae in the control group and the bacterial agent group on the 3rd day were 84.95% and 94.20%, respectively, and their survival rates on the 15th day were 100.97% and 132.61% (because the larvae budded). Since the coral larvae after attachment metamorphosis began to bud and reproduce, the survival rate of the bacterial agent group on the 15th day was greater than that of the control group; at the same time, Figure 3 As shown in (B), the budding rate of coral larvae in the inoculant group was higher than that in the control group. The results showed that the addition of inoculants can increase the survival rate of coral larvae and promote the budding reproduction of coral larvae.

[0045] The present invention comprehensively analyzes the effects of the addition of bacterial agents on the 24h attachment rate of coral planula larvae, the metamorphosis rate of planula larvae, the survival of coral larvae and their budding reproduction. The results show that the composite bacterial agent prepared by the combination of Phytobacter sp. SCSIO43973 and Salipiger sp. SCSIO43974 strains isolated and obtained from the coral reef ecosystem can significantly improve the 24h attachment rate, 72h metamorphosis rate and the extension tentacle ratio of planula larvae, and promote the survival and budding reproduction of coral larvae. These effects help to improve the replenishment rate of new coral individuals and promote the restoration of coral reef ecosystems. The composite bacterial agent provided by the present invention, as an effective microbial resource, has important scientific value and practical application significance for marine ecological protection and coral reef restoration. By utilizing these specific microbial strains, the attachment and metamorphosis process of coral larvae can be enhanced, and their survival rate in the natural environment can be improved, which is very important 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. Phytobacter sp. SCSIO43973, deposit number: GDMCC NO: 65583.

2. Salipiger sp.SCSIO43974, deposit number: GDMCC NO:65650.

3. A composite microbial agent that efficiently promotes the attachment, metamorphosis and larval growth of coral larvae, characterized in that: It comprises the Phytobacter sp. SCSIO43973 described in claim 1, the Salipiger sp. SCSIO43974 described in claim 2, or a combination thereof.

4. The composite microbial agent according to claim 3, characterized in that: The coral larvae and juveniles include but are not limited to Acropora hornba larvae and juveniles.

5. The composite microbial agent according to claim 3, characterized in that: The composition contains Phytobacter sp. A microbial inoculant prepared by mixing the bacterial solution of SCSIO43973 and the bacterial solution of Salipiger sp. SCSIO43974 in a volume ratio of 1:

1.

6. Phytobacter sp.SCSIO43973 described in claim 1, Salipiger sp. described in claim 2. Application of SCSIO43974 or a combination thereof in increasing the attachment and metamorphosis rate of coral larvae and promoting the growth of coral larvae.

7. The use according to claim 6, characterized in that: The composition contains Phytobacter sp. A microbial inoculant prepared by mixing the bacterial solution of SCSIO43973 and the bacterial solution of Salipiger sp. SCSIO43974 in a volume ratio of 1:

1.

8. A method for increasing the attachment and metamorphosis rate of coral larvae and promoting the growth of coral larvae, characterized in that: The method is to release the Phytobacter sp. SCSIO43973 described in claim 1, the Salipiger sp. SCSIO43974 described in claim 2 or a combination thereof into a culture system of coral larvae.

9. The method according to claim 8, characterized in that The coral larvae and coral juveniles include but are not limited to Acropora hornba larvae and juveniles.

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