Bacillus firmus gxu-z9 for preventing and treating coral bacterial bleaching and application thereof

Abstract

The application discloses a bacillus firmus GXU-Z9 for preventing and treating coral bacterial whitening, the bacillus firmus GXU-Z9 has a nucleotide sequence shown in SEQ ID NO:1, and is used for preventing and treating the whitening of antipathella japonica caused by pathogenic bacteria Vibrio coralliilyticus The bacillus firmus GXU-Z9 is obtained through antagonistic experiment screening, and then is identified through PCR and 16S rRNA gene sequencing. The bacillus firmus GXU-Z9 obtained through screening can antagonize the coral pathogenic bacteria Vibrio coralliilyticus Cause the whitening of coral, is applied to the prevention of specific diseases of coral, has great significance to the protection of coral reef resources and the improvement of the survival rate of artificial coral transplantation, and has certain positive effects on helping China to achieve long-term development of coral reef ecosystem protection.

Description

Technical Field

[0001] This invention belongs to the field of coral disease prevention and control technology, and specifically relates to a robust Bacillus GXU-Z9 for preventing and controlling bacterial bleaching of corals and its application. Background Technology

[0002] Coral reef ecosystems are among the most productive marine ecosystems, possessing extremely high biodiversity and significant economic value. However, in recent decades, coral reefs have gradually degraded. According to a survey by Mulhall M et al., from the early 1980s to 2000, the total area of ​​coral reefs decreased from approximately 600,000 square kilometers to approximately 250,000 square kilometers. Many factors contribute to coral reef degradation, including natural environmental factors (climate change, ocean acidification, El Niño, bleaching, cyclones), anthropogenic factors (explosive fishing, overuse of biological resources, farmland runoff, chemical pollution), and biological factors (starfish feeding, competition from macroalgae, pathogenic microorganisms). Among these, pathogenic microorganisms are a major cause of coral degradation. Due to the influence of environmental and anthropogenic factors, corals' immunity declines, further making them more susceptible to infection and damage from pathogens. To date, coral diseases caused by pathogenic microorganisms have been found to affect 106 coral species across 54 countries.

[0003] In 2002, Y. Ben-Haim et al. isolated from diseased staghorn cup corals... Vibrio coralliilyticus And it was verified through Koch's theorem. Vibrio coralliilyticus It is the pathogen of staghorn cup coral. Vibrio coralliilyticus It is a temperature-dependent pathogen that can cause the lysis of zooxanthellae and tissue loss in staghorn cup corals at temperatures above 27°C, leading to coral bleaching. Since the 21st century, Vibrio coralliilyticus Coral bleaching events are frequent, therefore, exploration... Vibrio coralliilyticus Prevention and control of bleaching caused by staghorn cup corals is of particular importance.

[0004] Currently, the main methods used for the prevention and treatment of coral diseases include physical methods, antibiotic therapy, probiotic therapy, and phage therapy. Among these, probiotic therapy is the primary method discussed in this invention, and its application in the prevention and treatment of aquatic organism diseases has been confirmed and verified by many scholars. Zhu Zhengxiang et al. found that adding an appropriate concentration of Clostridium butyricum to feed can improve the immunity of carp, regulate the intestinal flora structure, and inhibit the proliferation of harmful bacteria; Gu Wu et al. found that the probiotic spores they obtained have beneficial effects on the growth, development, and disease resistance of shrimp, and enhance the ability of Litopenaeus vannamei to resist Vibrio parahaemolyticus infection. In recent years, probiotic therapy has also been shown to have certain effects in the prevention and treatment of coral diseases. Philippe M. Rosado et al. found that using probiotic colonies can alleviate... Vibrio coralliilyticus This leads to coral bleaching. However, for Vibrio coralliilyticus There is currently no effective method to prevent or control bleaching of staghorn cup corals caused by this condition. Summary of the Invention

[0005] The purpose of this invention is to provide a robust Bacillus strain for preventing and treating bacterial bleaching of corals and its application in preventing and treating coral diseases caused by pathogenic bacteria.

[0006] The *Bacillus stolonifer* strain that prevents bacterial bleaching of corals has the nucleotide sequence shown in SEQ ID NO: 1 and is named *Bacillus stolonifer* GXU-Z9.

[0007] The *Bacillus stolonifer* GXU-Z9 described herein is deposited at the China Center for Type Culture Collection (CCTCCNO):2024727.

[0008] Based on whole-genome sequencing analysis of the probiotic characteristics of Bacillus stolonifer GXU-Z9, it was found that the chromosome genome of Bacillus stolonifer GXU-Z9 is a single-stranded circular genome, capable of encoding and synthesizing nutrients including various amino acids, vitamins, and polysaccharides, providing essential nutrients for coral growth and development; it can synthesize enzymes beneficial to corals, mainly including chitinase, β-glucosidase, and three antioxidant enzymes, helping corals digest organic particles and alleviate oxidative damage; it has the potential to produce penicillin and bacteriocins, which are beneficial for resisting the colonization and growth of pathogens; and it encodes three proteins related to immune regulation.

[0009] This invention, through genomic analysis, discovered that *Bacillus stolonifer* GXU-Z9 possesses the ability to synthesize seven amino acids, most of which are essential amino acids. *Bacillus stolonifer* GXU-Z9 may provide essential amino acids for its coral host. Furthermore, *Bacillus stolonifer* GXU-Z9 also has the ability to synthesize five B vitamins. B vitamins are important cofactors for enzymes and coenzymes required to maintain cellular metabolism. PoilloporaThe host genome of corals lacks the ability to synthesize B vitamins. Folic acid, an essential nutrient for nucleotide synthesis and methyl donation reactions, plays a crucial role in the growth, development, and reproduction of organisms. Riboflavin exists primarily as a coenzyme in the forms of flavin adenine dinucleotide and flavin mononucleotide, enhancing the activity of antioxidant enzymes and alleviating oxidative stress. Pantothenic acid is a precursor to coenzyme A (CoA), an essential cofactor for energy metabolism and fatty acid oxidation; studies have found that pantothenic acid and CoA have immunostimulatory effects. Thiamine is a cofactor for several enzymes involved in energy metabolism; research has shown that thiamine can enhance plant resistance to pathogens and activate plant defense systems. Cobalamin production has been proposed as one of the potential mechanisms for coral probiotics; its biosynthesis involves several metabolic pathways, including the production of glutathione and DMSP antioxidants. Corals and their symbiotic algae cannot synthesize cobalamin and must obtain it from symbiotic bacteria. Therefore, several B vitamins produced by *Bacillus stolonifera* GXU-Z9 may provide a nutritional source for corals during periods of stress, reducing oxidative stress and enhancing their resistance. Coral larvae can utilize trehalose provided by the symbiotic bacteria during the initial developmental stage of symbiosis and continue into adulthood. Thus, the trehalose produced by the coral symbiotic bacteria can promote the establishment of the symbiotic relationship, which can also be considered one of the probiotic properties of *Bacillus stolonifera* GXU-Z9.

[0010] The application of the aforementioned Bacillus stolonifer GXU-Z9 for preventing and controlling bacterial bleaching of coral is used to protect against bacterial bleaching of coral caused by pathogens of staghorn cup coral.

[0011] Preferably, the *Bacillus stolonifer* GXU-Z9 enhances the resistance of staghorn cup corals to bacterial bleaching by synthesizing pantothenic acid and indirectly promoting the synthesis of N-α-acetyl-L-lysine.

[0012] Preferably, the final concentration of the *Bacillus stolonifer* GXU-Z9 in the staghorn cup coral body is 4.00 × 10⁻⁶. 5 ~6.00×10 5 CFU / mL.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention, through antagonistic experiments, screened and found that Bacillus stolonifer GXU-Z9 can antagonize coral pathogens. Vibrio coralliilyticus Targeting pathogenic bacteria Vibrio coralliilyticus Coral bacterial bleaching caused by this pathogen can be controlled by adding a certain concentration of Bacillus thuringiensis GXU-Z9. The results of the control can be verified by analyzing the coral's appearance characteristics, and the test results are rapid and reliable.

[0014] 2. The probiotic screening and identification method and prevention and control method of the present invention are scientific and reliable, low in cost, simple to operate, highly efficient in prevention and control and have a high sample survival rate. They can be operated on a large scale and can be applied to the prevention of specific coral diseases. They are of great significance for protecting coral reef resources and improving the survival rate of artificial coral transplantation. They also play a positive role in helping my country’s coral reef ecosystem protection achieve long-term development. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the plate screening results of antagonistic probiotics in Example 1.

[0016] Figure 2 The appearance characteristics of staghorn cup-shaped corals in different treatment groups in Example 2.

[0017] Figure 3 The density of zooxanthellae symbiotic with staghorn cup corals in different treatment groups in Example 2.

[0018] Figure 4 The Fv / Fm values ​​of the staghorn cup corals in different treatment groups in Example 2 are shown.

[0019] Figure 5 The appearance characteristics of staghorn cup-shaped corals in different treatment groups in Example 3. Detailed Implementation

[0020] The present invention will be further described below with reference to the accompanying drawings. In the embodiments, unless otherwise specified, the technical means used are all conventional techniques in the art. The coral pathogen described in the embodiments is *Vibrio solani*. Vibrio coralliilyticus The strain was deposited at the Marine Microbial Culture Collection Center, accession number MCCC 1A00545, and was obtained through commercial sales. Example 1: Isolation, purification, classification and identification of Bacillus stolonifera GXU-Z9, which controls coral pathogens.

[0021] This embodiment provides a probiotic for preventing bacterial bleaching of corals. The probiotic is Bacillus firmus, which has the nucleotide sequence described in SEQ ID NO: 1 and is named Bacillus firmus GXU-Z9. It was deposited at the China Center for Type Culture Collection on April 19, 2024, with accession number CCTCC NO: M2024727.

[0022] The specific isolation and purification process of the Bacillus thymotherum GXU-Z9 is as follows: (a1) Sample selection: Healthy staghorn cup corals in good condition were collected from the Luhuitou Reef (109°28′E, 18°13′N) in the western part of Luhuitou Peninsula in southern Hainan Island and placed in the culture pond for 20 days to adapt.

[0023] Based on the growth characteristics of corals, the water quality conditions of the culture pond were set as follows: T: 29℃, KH: 7.0±0.3, pH: 8.2±0.1, Ca2+: 400±15ppm, Mg2+: 1400±20ppm, PO43-<0.03ppm, NH3+<0.15ppm, NO2-<0.1ppm, NO3- ≈0ppm. A 12 / 12h light cycle was set, using a 250W Metal halogen lamp + 4 T5HO, ODYSSEA, and newly hatched brine shrimp were fed twice a week.

[0024] (a2) Strain culture: The strains to be screened and coral pathogenic strains ( Vibrio coralliilyticus The strains were inoculated into MA liquid medium and cultured at 29°C and 160 rpm. The OD value of the strains was measured and their growth curves were plotted. The strains to be screened were isolated and cultured from corals and seawater, and the pathogenic bacteria were purchased from the National Microbial Resource Platform (NIMR).

[0025] (a3) Antagonistic screening experiment: (a31) Target strain application: When the coral pathogenic strain ( Vibrio coralliilyticus When the culture reaches the stationary phase in MA liquid medium, transfer 200 μL of the bacterial suspension and spread it onto MA solid medium. At the same time, attach antimicrobial susceptibility testing discs containing the strain to be screened onto the MA solid medium containing the coral pathogenic strain.

[0026] (a32) Strain culture: Place the plates from step (a31) in a 29°C incubator and observe for the appearance of inhibition zones. When inhibition zones appear, take a photograph and record the results. The results are as follows: Figure 1 As shown, two bacteria on the plate exhibited distinct inhibition zones.

[0027] (a4) Strain identification: When a clear inhibition zone appeared around the antimicrobial susceptibility test strip on the MA plate, the corresponding screening strain was subjected to PCR and 16S rRNA gene sequencing to identify its species. Finally, one of them was selected as the target potential probiotic. The selected probiotic has the nucleotide sequence shown in SEQ ID NO: 1 and was named Bacillus stoichiometrica GXU-Z9. Example 2: Application of Bacillus stolonifer GXU-Z9 in antagonizing pathogens of staghorn calamari.

[0028] In this embodiment, the *Bacillus thymotherum* GXU-Z9 obtained in Example 1 was used to treat the target pathogenic strain ( Vibrio coralliilyticus The prevention and control of bacterial bleaching of corals includes the following steps: (b1) Culture of strains: The selected Bacillus sturdierus GXU-Z9 and coral pathogens were added to MA medium and cultured until the stable phase.

[0029] (b2) Group processing: (b21) Coral grouping: The corals were divided into six groups of three, and the treatments were as follows: seawater was added alone as a blank group, pathogenic bacteria were added alone as a positive control group, Bacillus thymotherum GXU-Z9 was added alone as a negative control group, pathogenic bacteria and Bacillus thymotherum GXU-Z9 were added at the same time as experimental group 1, Bacillus thymotherum GXU-Z9 was added 5 hours in advance and then pathogenic bacteria were added as experimental group 2, and Bacillus thymotherum GXU-Z9 was added 10 hours in advance and then pathogenic bacteria were added as experimental group 3. (b22) Strain Treatment: When the strain reached the stationary phase, 18 mL of the target Bacillus stolonifer GXU-Z9 bacterial suspension was taken, centrifuged at 8000 rpm for 3 min, the supernatant was discarded, and the bacterial cells were resuspended in seawater to 6 mL. The resuspended bacterial suspension was added to 1 mL for each coral in experimental group 1 and the negative control group. Simultaneously, 1 mL of seawater was added to each coral in the blank control group. After 5 h, 9 mL of Bacillus stolonifer GXU-Z9 bacterial suspension was taken, centrifuged at 8000 rpm for 3 min, the supernatant was discarded, and the bacterial cells were resuspended in seawater to 3 mL. The resuspended bacterial suspension was added to 1 mL for each coral in experimental group 2. After 10 h, 9 mL of Bacillus stolonifer GXU-Z9 bacterial suspension was taken, centrifuged at 8000 rpm for 3 min, the supernatant was discarded, and the bacterial cells were resuspended in seawater to 3 mL. The resuspended bacterial suspension was added to 1 mL for each coral in experimental group 3. Additionally, the coral pathogenic bacteria (…) Vibrio coralliilyticus 6.6 mL of bacterial suspension was centrifuged at 8000 rpm for 3 min, the supernatant was discarded, and the bacterial cells were resuspended in seawater to a final volume of 2.2 mL. The resuspended bacterial suspension was then added to 180 μL for each coral in experimental groups 1, 2, 3, and the positive control group, to achieve a final concentration of *Bacillus stolonifer* GXU-Z9 in the staghorn cup coral water body of 4.00 × 10⁻⁶. 5 CFU / mL.

[0030] (b31) Inoculation observation: Take 18 beakers, place one coral in each beaker, and pour the strain and seawater from step (b22) onto the surface of the coral in chronological order. Then transfer it into the beaker, add 500 mL of seawater from the coral tank into the beaker, and put the coral back into the tank after 24 hours.

[0031] (b32) Data collection: During the experiment in (b31), the corals were photographed and recorded for later analysis of changes in the appearance characteristics of the corals under different conditions. The Fv / Fm value of chlorophyll fluorescence of symbiotic zooxanthellae was measured using a Diving-PAM underwater fluorometer (WALZ, Germany). The tissue fluid obtained by grinding the corals was filtered with filter paper to remove impurities, and the filtrate was counted under a microscope to calculate the zooxanthellae density. Finally, a control assessment was conducted.

[0032] (b4) Prevention and control assessment: like Figure 2 As shown, under the temperature condition of 29 ± 0.5℃, compared with before the experiment, the coral samples of the blank control group, the group inoculated only with Bacillus stolonifer GXU-Z9, and the experimental groups inoculated with Bacillus stolonifer GXU-Z9 5 h and 10 h in advance all showed a healthy state. The polyp tentacles were stretched, the color was normal, the tissues were intact, and there were no signs of bleaching. Simultaneous inoculation with Bacillus stolonifer GXU-Z9 and pathogenic bacteria... V. coralliilyticus Some corals showed slight shrinkage of individual polyps, but their color remained largely unchanged, and the tissues remained plump without obvious bleaching; while only inoculated with pathogenic bacteria... V. coralliilyticus All the corals exhibited polyp shrinkage, accompanied by severe tissue loss and significant bleaching. This demonstrates that, compared to simply inoculating with pathogens... V. coralliilyticus For the corals in the other treatment groups, the coral phenotypes were relatively healthy, indicating that Bacillus stolonifer GXU-Z9 effectively inhibits pathogenic bacteria. V. coralliilyticus It has a good mitigating effect on bacterial bleaching of staghorn cup corals.

[0033] exist Figure 2 In the figure, C represents the zooxanthellae content of coral samples in the blank control group, BO represents the zooxanthellae content of coral samples in the negative control group, V represents the zooxanthellae content of coral samples in the positive control group, VOS represents the zooxanthellae content of coral samples in experimental group 1, VOF represents the zooxanthellae content of coral samples in experimental group 2, and VOT represents the zooxanthellae content of coral samples in experimental group 3. As shown in the figure, VOT, VOF, C, and BO are significantly higher than V and VOS. This indicates that Bacillus stolonifera GXU-Z9 itself is non-toxic to coral samples, and adding Bacillus stolonifera GXU-Z9 to coral strains 5 or 10 hours in advance can effectively reduce the toxicity of coral pathogenic strains to coral zooxanthellae.

[0034] exist Figure 4In the figure, C represents the Fv / Fm value of coral samples in the blank control group, BO represents the Fv / Fm value of coral samples in the negative control group, V represents the Fv / Fm value of coral samples in the positive control group, VOS represents the Fv / Fm value of coral samples in experimental group 1, VOF represents the Fv / Fm value of coral samples in experimental group 2, and VOT represents the Fv / Fm value of coral samples in experimental group 2. As shown in the figure, by day 10 of the experiment, VOT, VOF, C, and BO began to be higher than V and VOS. This indicates that Bacillus stolonifera GXU-Z9 itself has little impact on photosynthetic efficiency, and that adding Bacillus stolonifera GXU-Z9 to the coral strains 5 h and 10 h in advance can effectively reduce the impact of pathogenic coral strains on coral photosynthetic efficiency.

[0035] The above results indicate that the method of preventing and controlling pathogenic bacteria by adding a certain concentration of Bacillus stolonifer GXU-Z9 is scientific and reliable, and can be applied to the prevention of specific coral diseases. It is of great significance for protecting coral reef resources and improving the survival rate of artificial coral transplantation, and plays a positive role in helping my country's coral reef ecosystem protection achieve long-term development. Example 3: Application of Bacillus thymotherum GXU-Z9 in the verification of its in vitro metabolites and antibacterial activity.

[0036] (a) Analysis of differences in metabolites.

[0037] The Venn diagram analysis was used to identify common differential metabolites in four probiotic GXU-Z9 inoculation groups (groups with Bacillus stolonifera GXU-Z9 alone, group with both pathogenic bacteria and Bacillus stolonifera GXU-Z9, group with Bacillus stolonifera GXU-Z9 added 5 hours in advance followed by pathogenic bacteria, and group with Bacillus stolonifera GXU-Z9 added 10 hours in advance followed by pathogenic bacteria) compared to the blank control group and the pathogenic bacteria inoculation group in Example 2.

[0038] The results showed that the four groups inoculated with probiotic GXU-Z9 shared five common differential metabolites compared with the blank control group, including two significantly upregulated metabolites: N-α-acetyl-L-lysine and laminine; and three significantly downregulated metabolites: 12-hydroxydodecanoic acid, (11Z,14Z)-3-Icosa-11,14-dienoylcarnitine and 4-methyl-5-thiazolylethanol.

[0039] The four groups inoculated with the probiotic GXU-Z9 shared 18 common metabolites compared to the group inoculated with only pathogenic bacteria, including 14 significantly upregulated metabolites: curcumin, glutamate leucine ester, 1-(2-Furyl)butan-3-one, tyramine, N-α-acetyl-L-lysine, 4-methylbenzaldehyde, proline dimethylolamine, butyryl-L-carnitine, phenol, cinnamyl acetate, Cer(d18:1 / 17:0), hemolysin, platelet-activating factor C-16, and 11-hydroxynonadenylate. The base, (5Z,8Z,13E,15S)-11,12,15-Trihydroxyicosa-5,8,13-trienoylcarnitine; and four significantly downregulated metabolites: 2-hydroxydodecanoic acid, (11Z,14Z)-3-Icosa-11,14-dienoylcarnitine, PC(2:0 / 18:1(12Z)-2OH(9,10)), and PC(18:1(12Z)-2OH(9,10) / 2:0). Furthermore, N-α-acetyl-L-lysine was significantly upregulated in corals inoculated with the probiotic GXU-Z9.

[0040] MIMOSA2 analysis, combining microbiome and metabolome data from six treatment groups, identified eight related [factors / elements]. Bacillus firmus DS1 has directly associated metabolites, including six positively correlated metabolites: L-proline, L-glutamine, hypoxanthine, sucrose, pantothenic acid, and xanthine; and two negatively correlated metabolites: betaine and glucosamine. Pantothenic acid was found to be associated with co-inoculation of *Bacillus stolonifer* GXU-Z9 and pathogenic bacteria. V. coralliilyticus The abundance of Bacillus stomatitis in the group that was inoculated with Bacillus stomatitis GXU-Z9 5 hours in advance and the group that was inoculated with Bacillus stomatitis GXU-Z9 10 hours in advance was significantly higher than that inoculated with pathogenic bacteria only. V. coralliilyticus Group; (ii) Validation of antibacterial activity of high-abundance metabolites.

[0041] The disease resistance results of the metabolites were verified by adding different metabolites at a temperature of 29±0.5℃. The experiment was divided into four groups: a control group with seawater added alone; a control group with only pathogenic bacteria added; and a control group with only pathogenic bacteria added. V. coralliilyticus Group 1; Pantothenic acid inoculation disease resistance group; N-α-acetyl-L-lysine inoculation disease resistance group; Corals in each group were photographed and observed for 7 days, and the results are as follows: Figure 4 As shown.

[0042] Depend on Figure 5As can be seen, the coral polyps in the blank control group showed normal extension, without tissue loss or bleaching; the corals in the pantothenic acid resistance group and the N-α-acetyl-L-lysine resistance group also showed normal polyp extension, without tissue loss or bleaching; only inoculated with pathogenic bacteria V. coralliilyticus The corals exhibited polyp shrinkage, accompanied by severe tissue loss and bleaching. This demonstrates that pantothenic acid and N-Alpha-acetyllysine have the best resistance to bleaching of staghorn cup corals caused by the pathogenic bacterium *V. coralliilyticus*. The above embodiments are merely exemplary embodiments of the present invention and are not intended to limit the present invention. The scope of protection of the present invention is defined by the claims. Those skilled in the art can make various modifications or equivalent substitutions to the present invention within its spirit and scope of protection, and such modifications or equivalent substitutions should also be considered to fall within the scope of protection of the present invention.

Claims

1. A robust Bacillus GXU-Z9 strain for preventing and controlling bacterial bleaching of corals, characterized in that: The described Bacillus thymotherum GXU-Z9 has the nucleotide sequence shown in SEQ ID NO:

1.

2. The Bacillus thuringiensis GXU-Z9 for preventing and controlling coral bacterial bleaching according to claim 1, characterized in that: The aforementioned Bacillus thymotherum GXU-Z9 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:2024727.

3. The application of the robust Bacillus GXU-Z9 for preventing and controlling coral bacterial bleaching as described in claim 1, characterized in that: Used for the prevention and treatment of bacterial bleaching of corals caused by pathogens of staghorn cup coral.

4. The application of the Bacillus thuringiensis GXU-Z9 for preventing and controlling coral bacterial bleaching according to claim 3, characterized in that: The aforementioned Bacillus stolonifer GXU-Z9 enhances the resistance of staghorn cup corals to bacterial bleaching by synthesizing pantothenic acid and indirectly promoting the synthesis of N-α-acetyl-L-lysine.

5. The application of the Bacillus thuringiensis GXU-Z9 for preventing and controlling bacterial bleaching of corals according to claim 4, characterized in that: The final concentration of the *Bacillus stolonifer* GXU-Z9 in the staghorn cup coral body was 4.00 × 10⁻⁶. 5 ~6.00×10 5 CFU / mL.

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