Flavobacteria bacterium with efficient promotion effect on coral larva attachment and metamorphosis and application of flavobacteria bacterium

Abstract

The invention discloses a flavobacteria bacterium capable of efficiently promoting attachment and metamorphosis of coral larvae and application of the flavobacteria bacterium. The Flavobacterium sp. SCSIO 2518 is preserved in the Guangdong Microbial Culture Collection Center on November 14, 2025, the address is Courtyard 100, Xianlie Middle Road, Guangzhou, the postcode is 510070, and the preservation number is GDMCC No.67297. The Flavobacterium sp. SCSIO 2518 is preserved in the Guangdong Microbial Culture Collection Center on November 14, 2025. The flavobacteria bacterium 2518 is separated and purified from calcified algae collected in a coral reef area of deer in trianya in Hainan province in China, can remarkably improve the adhesion rate and the metamorphosis rate of madrepore larvae and shorten time, and is free of toxic and side effects. Therefore, the method can be applied to promoting attachment and metamorphosis of the coral larvae, promoting artificial large-scale breeding of the corals and providing high-quality seedling resources for coral population recovery, so that restoration of a degenerated coral reef ecological system is promoted.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microorganisms, and particularly relates to a Flavobacteriaceae strain for promoting coral larva attachment metamorphosis and application thereof. BACKGROUND

[0002] Coral reef ecosystems are one of the most biologically diverse ecosystems in the world, providing not only survival (including habitat, foraging, and reproduction) for about 25% of marine species, but also coastal protection, food, and economic (fishery, tourism, etc.) sources for about 1 billion people worldwide. In addition, the rich biological resources in coral reef ecosystems also provide great potential for the development of new drugs and other products. However, in recent decades, global coral reef ecosystems have been severely degraded due to climate change and human activities (overfishing, pollution, etc.). In particular, the global warming-induced marine heat wave has caused multiple large-scale scleractinian coral bleaching events. As of 2025, 84% of global coral reef ecosystems have been affected by bleaching events, covering at least 82 countries and regions. There is an urgent need to study how to promote the recovery of scleractinian coral populations and thus accelerate the recovery of coral reef ecosystems.

[0003] Larval attachment and metamorphosis are the core bottleneck link in the life history of scleractinian corals, and their success rate directly affects the efficiency of juvenile replenishment and spatial distribution pattern, which is a prerequisite for the effective recovery of scleractinian coral populations. However, this critical process is highly dependent on exogenous induction signals: in the natural environment, if specific induction signals are lacking, larvae will not only significantly prolong the planktonic period, but also completely stop attachment, ultimately leading to the failure of juvenile replenishment, which is a core obstacle in the current recovery process of coral reef ecosystems. Therefore, it is crucial to develop methods that can efficiently improve the attachment and metamorphosis rate of coral larvae to accelerate the recovery of coral populations and the restoration of coral reef ecosystems.

[0004] Previous studies have shown that Pseudoalteromonas has the ability to induce metamorphosis of coral larvae, and its biofilm or metabolite tetra-bromopyrrole can efficiently promote the metamorphosis of coral larvae, but the effect of increasing the attachment rate is limited, which limits the application of this strain in coral reef restoration practice. In recent years, with the development of high-throughput sequencing technology, through the correlation analysis of bacterial abundance and coral larvae attachment metamorphosis rate, it is found that there are a large number of potential coral larvae attachment metamorphosis inducing bacteria resources in the natural environment. However, the research on these potential microbial resources has obvious shortcomings - only a few strains of Thalassomonas, Roseivivax, Pseudovibrio, Acinetobacter and Microbulbifer, Bacillus, Phytobacter, Salipiger have been confirmed to increase the attachment metamorphosis rate of coral larvae, and the functions of most potential inducing bacteria have not been verified, not to mention being transformed into practical application technology resources. Therefore, screening more strains with high efficient induction effect on coral larvae attachment metamorphosis not only can enrich the coral larvae inducing bacteria resource library, but also can provide key technical support for breaking the juvenile supplement bottleneck, promoting coral population restoration and coral reef ecosystem restoration, which has important theoretical research value and practical guiding significance. SUMMARY

[0005] The first object of the present application is to provide a Flavobacteriaceae SCSIO 2518 which can significantly increase the attachment rate and metamorphosis rate of coral larvae. The strain is isolated from calcified algae collected in Sanya Luhuitou sea area, Hainan Province, China. The strain was deposited with the Guangdong Microbial Culture Collection Center (GDMCC) on November 14, 2025, located at 100 Xianlie Road, Guangzhou, China, postcode: 510070, and the deposit number is GDMCC No. 67297.

[0006] Through the screening experiment of the inducing effect of bacteria on coral larvae, the results show that the bacterial liquid (concentration 10 μL bacterial liquid / mL seawater) of Flavobacteriaceae SCSIO 2518 fermented for 48 h can significantly promote the metamorphosis of coral larvae. After 96 h of induction, the attachment rate of coral larvae is 63.3%, and the metamorphosis rate (the metamorphosis rate mentioned in this paper is the attachment metamorphosis + metamorphosis not attached) is 78.3%.

[0007] The growth curve of Flavobacteriaceae SCSIO 2518 was determined and fitted by logistic model. The results show that the maximum growth rate of the strain is 0.0497 (OD 600Flavobacteriaceae SCSIO 2518 cells in the logarithmic phase (approximately 23 hours) entered the logarithmic phase at 23 hours and reached the stationary phase at 46 hours. Flow cytometry was used to count the cytosmic cells in the logarithmic phase, and the counts were compared with the corresponding OD values. 600 A linear regression model was established to obtain the relationship between the bacterial density and OD. 600 The correspondence between the values ​​is: y = 5,210,817,359.51x - 515,470,930.18 (R² = 1.00); where x represents OD. 600 y represents bacterial density (cells / mL).

[0008] The induction effect of Flavobacteriaceae SCSIO 2518 on coral larval attachment and metamorphosis at different stages was verified. The results showed that the logarithmic stage had the best induction effect. Further verification of the induction effect of Flavobacteriaceae SCSIO 2518 at different cell concentrations during the logarithmic stage revealed that the optimal induction concentration for Flavobacteriaceae SCSIO 2518 was 4 × 10⁻⁶. 7 At this concentration of cells / mL, after 96 h of induction, the attachment rate of coral larvae was 48.3% and the metamorphosis rate was 75%.

[0009] The second objective of this invention is to provide a microbial preparation that promotes the attachment and metamorphosis of coral larvae, containing Flavobacteriaceae SCSIO 2518 cells or its active ingredients, which can be applied to the artificial breeding of corals and the ecological restoration of coral reefs.

[0010] Preferably, the coral larvae are larvae of the staghorn cup coral (Pocillopora damicornis).

[0011] Preferably, the Flavobacteriaceae SCSIO 2518 cells are in the logarithmic growth phase.

[0012] Further optimization involves placing the staghorn cup coral larvae in a solution containing a concentration of 4×10 7 Flavobacteriaceae SCSIO 2518 filters seawater at cells / mL.

[0013] The larval density of staghorn cup corals mentioned above is 1 larva / mL of seawater.

[0014] The Flavobacteriaceae SCSIO 2518 is obtained by separation and purification from calcified algae collected in the Sanya Luhuitou Coral Reef Area in Hainan Province, China, and the bacterium can significantly improve the attachment rate and metamorphosis rate of coral larvae, shorten the time, and has no toxic side effects. Therefore, the present application can be applied to promote coral larvae attachment and metamorphosis, promote artificial large-scale breeding of corals, provide high-quality seed resources for coral population recovery, and thus promote the repair of degraded coral reef ecosystems.

[0015] Flavobacteriaceae SCSIO 2518 was preserved in Guangdong Microbial Culture Collection Center (GDMCC) on November 14, 2025, at address: 100, Xianlie Middle Road, Guangzhou, China, postcode: 510070, preservation number: GDMCC No. 67297. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is a phylogenetic tree of Flavobacteriaceae SCSIO 2518;

[0017] Figure 2 is an induction effect diagram of Flavobacteriaceae SCSIO 2518 bacterial solution in the screening experiment;

[0018] Figure 3 is the growth curve and logarithmic phase OD of Flavobacteriaceae SCSIO 2518; 600 bacterial density linear model;

[0019] Figure 4 is the effect of different concentrations of Flavobacteriaceae SCSIO 2518 on the attachment and metamorphosis of Pocillopora damicornis larvae. DETAILED DESCRIPTION

[0020] The following examples are specific introductions to the present application, but not limitations to the present application.

[0021] Example 1: Isolation and identification of strain Flavobacteriaceae SCSIO 2518

[0022] Strain isolation: Flavobacteriaceae SCSIO 2518 was isolated from Porolithon onkodes collected in the Sanya Luhuitou Coral Reef Area in December 2022, and the culture medium used was 2216E solid medium (Hibio).

[0023] Strain 16S rRNA sequence identification: Bacterial DNA was extracted using chelating resin Chelex 100. Specifically, a single colony of the strain was picked and inoculated into 100 μL of 10% Chelex 100, and then boiled in a water bath for 10 min. After centrifugation at 12,000 rpm for 10 min, the supernatant was taken as the DNA template for PCR amplification of the 16S rRNA gene. The forward and reverse primers were 27F (5'-AGAGTTTGATCCTGGCTCA-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'), respectively. The PCR reaction system was as follows: 2x EasyTaq SuperMix 25 μL, 1 μL of the forward and reverse primers, respectively, 1 μL of the DNA template, and 22 μL of ddH2O. The PCR reaction program was as follows: 95 °C pre-denaturation for 5 min, 95 °C denaturation for 30 s, 50 °C annealing for 45 s, 72 °C extension for 90 s, 30 cycles, and finally 72 °C extension for 10 min. The size and integrity of the PCR products were tested by agarose gel electrophoresis. The PCR products that met the requirements were sent to Tianyi Huiyuan Biotechnology Co., Ltd. (Guangdong, Guangzhou) for Sanger sequencing. The 16S rRNA sequence of Flavobacteriaceae SCSIO 2518 is shown as SEQ ID NO. 1. By comparing with the bacterial 16S rRNA sequences in the EzBioCloud database, the most similar effective published strain of Flavobacteriaceae SCSIO 2518 was Croceivirga thetidis (DJ-13(T) with a similarity of 93.43%), which was lower than the universal threshold (94.50%) for the classification of new bacterial genera (Chun et al., 2018, Proposed minimal standards for the use of genome data for the taxonomy of prokaryotes. International Journal of Systematic and Evolutionary Microbiology. 68: 461-466). Further, the corresponding 20 closest sequences with similar degrees were obtained from the database, and MEGA11 software was used for multiple sequence alignment, and a phylogenetic tree was constructed using the neighbor-joining method. The tree results of the strain Flavobacteriaceae SCSIO 2518 are shown in Figure 1. Figure 1As shown, it belongs to Bacteroidetes, Flavobacteriia, Flavobacteriales, Flavobacteriaceae; since the sequence similarity of the strain and its close strain is lower than 94.5%, and it is separately classified as a branch on the phylogenetic tree, it is preliminarily proved to be a potential new genus of Flavobacteriaceae, named Flavobacteriaceae SCSIO 2518, and preserved in Guangdong Microbial Culture Collection Center (GDMCC) on November 14, 2025, address: 100, Martyrs' Avenue, Guangzhou, China, postcode: 510070, preservation number: GDMCC No. 67297.

[0024] Example 2: Preliminary determination of the induction effect of strain Flavobacteriaceae SCSIO 2518

[0025] I. Activation of strain Flavobacteriaceae SCSIO 2518

[0026] The strain preservation tube was taken out, thawed at room temperature, and 50 μL was uniformly coated on the 2216E solid culture medium (Hibio), and placed in a constant temperature incubator at 28°C for 3 days. Single colonies were picked and streaked for purification.

[0027] II. Preliminary determination of the induction effect of strain Flavobacteriaceae SCSIO 2518

[0028] The purified bacteria were inoculated into 30 mL of 2216E liquid medium, and fermented at 28°C and 180 rpm for 48 h to obtain the strain fermentation broth (bacterial broth). The induction effect of the strain on the attachment metamorphosis of Pocillopora damicornis larvae was investigated. The attachment experiment was carried out in a sterile six-well plate. The experimental group was 10 coral larvae + 9.9 mL of sterilized filtered seawater (AFSW) + 100 μL of bacterial broth; the blank control group was 10 coral larvae + 9.9 mL of sterilized filtered seawater (AFSW) + 100 μL of 2216E liquid medium. Each group had 6 replicates. After 48 h or 96 h, the attachment metamorphosis and death of the larvae were observed and recorded under a dissecting microscope. The results are shown in Figure 2 As shown, after 48 h, the attachment rate of Pocillopora damicornis larvae was 31.7%, and the metamorphosis rate was 36.7%; after 96 h, the attachment rate of the larvae was 63.3%, and the metamorphosis rate was 78.3%, which was significantly higher than that of the blank control, indicating that it had the potential to efficiently promote the attachment metamorphosis of coral larvae.

[0029] Example 3: Growth curve determination of strain Flavobacteriaceae SCSIO 2518

[0030] I. Growth curve determination of strain Flavobacteriaceae SCSIO 2518

[0031] A loop was used to inoculate the activated and purified bacteria into 30 mL of 2216E liquid medium, and the seed liquid was prepared by fermentation at 28°C and 180 rpm for 24 h. The seed liquid was inoculated into 50 mL of fresh 2216E liquid medium (n = 3) at a 1:100 v / v inoculation amount (500 μL) and subjected to shaking culture at 28°C and 180 rpm. The bacterial suspension OD 600 values (n = 3) were determined using a microplate reader, with sterile 2216E medium as a blank control. The determination period of strain Flavobacteriaceae SCSIO 2518 was 72 h. The logistic model was used for fitting, where Asym is the maximum OD value, Xmid is the inflection point time of the logarithmic phase, and Scal is the scale parameter (negatively correlated with the steepness of the curve). The maximum growth rate (OD max / h) was calculated by the formula μ 600 = Asym / (4×Scal), and the growth lag phase was estimated according to the empirical formula Lag phase ≈ Xmid - 2×Scal. The growth curve of strain Flavobacteriaceae SCSIO 2518 is shown in FIG. A, and the fitted curve is Figure 3 According to the estimation, the lag phase of strain Flavobacteriaceae SCSIO 2518 was 23 h (i.e., after which the growth phase began), and it gradually entered the stationary phase at 46 h, with a maximum growth rate of 0.0497 (OD 600 / h).

[0032] II. Logarithmic phase cell density-OD 600 linear model drawing

[0033] ​At the sampling time points of the above growth curve measurement, an additional 1.96 mL of bacterial suspension was taken for bacterial density quantification (cells / mL). The steps are as follows: (1) bacterial liquid fixation: add 40 μL glutaraldehyde, fix the sample for 5 minutes, and store at -30°C after quick freezing in liquid nitrogen. (2) Cell counting: Take the logarithmic phase sample of the strain, thaw at room temperature, filter 1 mL with a 600-mesh silk screen, dilute the filtrate to the appropriate concentration with sterile ddH2O, add 10 μL of SYBR dye, and stain in the dark for 10 minutes. Then, use a flow cytometer to count the sample. Select the OD 600 values at the logarithmic phase time points (25-35 h, 38-39 h) and establish a linear regression model with the corresponding cell density, as shown in Figure 3 B. The corresponding relationship between the bacterial density of the strain Flavobacteriaceae SCSIO 2518 at the logarithmic phase and the OD 600 value is y = 5,210,817,359.51x - 515,470,930.18 (R² = 1.00); where x represents OD 600 , and y represents bacterial density (cells / mL).

[0034] Example 4: Determining the optimal induction concentration of the strain Flavobacteriaceae SCSIO 2518

[0035] The Dipsastraea cervicornis was collected from the Sanya Luhuitou sea area in Hainan Province, China in August 2024, and the larvae were collected and cultured in indoor 27°C sand-filtered seawater under aeration for standby use.

[0036] According to the growth curve, the logarithmic phase bacterial suspension of the strain Flavobacteriaceae SCSIO 2518 was centrifuged at 8000 rpm for 10 min at room temperature, and after discarding the supernatant, it was resuspended and diluted to 1×10 7 , 2×10 7 , 4×10 7 , 6×10 7 , 8×10 7 , 1×10 8 cells / mL for larval attachment experiments. The attachment experiment was carried out in a sterile six-well plate, and the experimental group was 10 coral larvae + 10 mL AFSW + corresponding gradient concentration of bacteria; the blank control group was 10 coral larvae + 10 mL AFSW. Each group had 6 replicates. The experimental light intensity was about 70 μmol photons m -2 s -1, light and dark cycle was 12h / 12h, and temperature was controlled at 27±1°C. During the period, metamorphosis and death of larvae were observed and recorded under a dissecting microscope at 48h and 96h; half of the seawater and bacteria in the system were replaced at 48h. The results of the induction experiment of strain Flavobacteriaceae SCSIO 2518 are shown in Table 1. Figure 4 After 48h, the attachment rate of the strain treatment group ranged from 0 to 16.7%, and the metamorphosis rate ranged from 0 to 45%, which had no statistical difference compared with the control group. However, after 96h, the attachment rate of the strain treatment group ranged from 1.7% to 48.3%, and the metamorphosis rate ranged from 3.3% to 75%, among which, the metamorphosis rates of the 4×10 7 and 8×10 7 cells / mL concentration groups were 75% and 66.7% respectively, which were significantly higher than that of the control group, and there was no death of larvae after 96h in the two groups. In summary, the optimal induction concentration of the bacteria was 4×10 7 cells / mL.

[0037] The above is only the preferred embodiment of the present application, and it should be noted that the above embodiment does not limit the present application, and any technical solution obtained by optimization and improvement on the basis of the above scheme does not exceed the protection scope of the present application.

[0038] 16S rRNA sequence of Flavobacteriaceae SCSIO 2518 (SEQ ID NO. 1)

[0039]

Claims

1. Flavobacteriaceae SCSIO 2518, with a preservation number of GDMCC No. 67297.

2. A microbial preparation capable of promoting settlement metamorphosis of coral planulae, characterized by, A microbial preparation containing the Flavobacteriaceae SCSIO 2518 strain or active ingredients therein.

3. Use of the Flavobacteriaceae SCSIO 2518 strain of claim 1 or the microbial preparation of claim 2 in promoting the settlement metamorphosis of coral larvae.

4. Use according to claim 3, characterized in that, The coral larvae are Pocillopora damicornis larvae.

5. Use according to claim 3, characterized in that, The Flavobacteriaceae SCSIO 2518 strain is a logarithmic growth phase strain.

6. Use according to claim 3, characterized in that, are placed in filtered seawater containing Flavobacteriaceae SCSIO 2518 at a concentration of 4 x 10 7 cells / mL. are placed in filtered seawater containing Flavobacteriaceae SCSIO 2518 at a concentration of 4 x 10 7 cells / mL.

7. Use according to claim 3, characterized in that, The density of the Pocillopora damicornis larvae is 1 larva / mL of seawater.

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