A strain of rhodobacter sphaeroides and its application in promoting the formation of sporangial branches of porphyra haitanensis

Abstract

The application provides a strain of pink bacillus and application of the strain in promoting formation of sporangiophores of porphyra haitanensis, and the preservation number of the pink bacillus is CCTCC NO: M 20242610. By adding the pink bacillus strain in a porphyra haitanensis filament culture system, development of sporangiophores can be effectively promoted, the proportion of sporangiophores can be significantly increased to 48.8% after 4 weeks of culture, directional maturation under artificial intervention is realized, the shortcomings of long cycle and poor stability of traditional environmental regulation means are overcome, and the consistency and quality of chlamydospore dissemination are improved, and a new technical approach is provided for porphyra haitanensis seedling production.

Description

Technical Field

[0001] This invention belongs to the field of filamentous seedling technology of laver, specifically involving a pink rod-shaped fungus and its application in promoting the formation of sporangial branches of laver. Background Technology

[0002] Pyropia haitanensis is an important economic seaweed endemic to my country, with its cultivation scale and yield ranking among the world's top. Artificial cultivation is a complex process, relying on the production of conchospores during the filamentous stage and their further development into visible thallus. Among these, the growth and development of the filamentous stage, especially the differentiation of enlarged algal filaments and the formation of sporangial branches, is a crucial link in the entire life cycle, directly determining the quantity and quality of conchospores, and thus affecting the yield and economic benefits of the seaweed.

[0003] In production practice, the development of filaments and the formation of sporangia are usually induced by regulating environmental factors. The main measures include: temperature regulation - simulating seasonal changes by changing water temperature to promote the formation of enlarged algal filaments and conchospores; light control - adjusting light intensity and photoperiod to affect photosynthesis and morphogenesis of filaments; and nutrient management - optimizing the concentration and ratio of elements such as nitrogen (N) and phosphorus (P) to provide nutritional support for the development of filaments.

[0004] However, these traditional methods have certain limitations: first, they are time-consuming, often requiring several weeks from filament culture to the formation of a large number of conchospores, thus limiting production efficiency; second, they lack stability, easily affected by external environmental fluctuations, leading to significant differences in sporulation effects between different batches, thereby threatening seedling supply; and third, they rely on macro-environmental control, making it difficult to precisely stimulate the inherent developmental potential of the filaments, limiting further improvement in the efficiency per unit of seedling water. Therefore, the industry urgently needs a more efficient, stable, and safe new technology to promote filament swelling and sporangial branch formation.

[0005] In recent years, the interaction between seaweed and microorganisms has gradually attracted attention. Complex algal microbial communities inhabit the surface of large seaweeds, the surrounding water, and the substrate they attach to. These microorganisms play a crucial role in the growth, development, and disease resistance of seaweeds by secreting signaling molecules, hormones, vitamins, or specific metabolites. Previous studies have shown that certain bacteria can promote the morphogenesis of the model algae *Ulva*. However, current research mainly focuses on the overall microbiome function or the growth effects at the thallus stage, with insufficient attention paid to the filamentous development stage of *Porphyra*. The resources of functional bacterial strains that can specifically and efficiently promote the swelling of *Porphyra* filaments and the formation of sporangia are extremely limited, and their industrial application is almost nonexistent.

[0006] Isolating functional strains that promote the development of filamentous structures in Porphyra yezoensis not only helps to reveal the algae-bacteria interaction mechanism, but also provides a new and environmentally friendly microbial regulation strategy for Porphyra yezoensis seedling cultivation, which is of great significance and has broad prospects for promoting the sustainable development of Porphyra yezoensis aquaculture. Summary of the Invention

[0007] The purpose of this invention is to provide a pink rod-shaped fungus and its application in promoting the formation of sporangial branches of Porphyra yezoensis. This strain can significantly promote the development of sporangial branches of Porphyra yezoensis filaments and shorten its maturation cycle, thereby solving the problem of long development cycle of Porphyra yezoensis filaments in the prior art.

[0008] The present invention first provides a pink rod-shaped bacterium (Roseivirga sp.) MT strain, which is deposited at the China Center for Type Culture Collection of Wuhan University, with accession number CCTCC NO: M 20242610 and deposit date of November 21, 2024.

[0009] The present invention also relates to the use of the strain in the preparation of formulations for promoting the development of filamentous sporangial branches of Porphyra yezoensis.

[0010] Furthermore, the preparation is a microbial bacterial solution.

[0011] The present invention also provides a microbial preparation comprising live bacteria of the aforementioned pink rod-shaped bacteria;

[0012] Furthermore, the microbial preparation also contains the fermentation supernatant of the pink rod-shaped bacteria.

[0013] The fermentation supernatant is prepared by inoculating the pink rod-shaped bacteria into 2216E liquid culture medium, activating and culturing it at 26℃ and 200r / min for 24 h, and then centrifuging it to obtain the supernatant.

[0014] Another objective of this invention is to provide a method for promoting the maturation of sporangial branches of *Porphyra yezoensis* filaments, wherein the method involves culturing free filaments of *Porphyra yezoensis* by adding the aforementioned pink rod-shaped fungus or its fermentation supernatant to the filament culture medium.

[0015] The cultivation conditions were: temperature 28℃, light intensity 500 Lx, and photoperiod 8L:16D.

[0016] This invention, by adding pink rod-shaped strains to the filamentous culture system of *Porphyra yezoensis*, can effectively promote the development of sporangial branches. After 4 weeks of culture, the proportion of sporangial branches formed can be significantly increased to 48.8% (compared to 18.3% in the control group). This achieves directional ripening under artificial intervention, overcoming the shortcomings of traditional environmental control methods, such as long cycles and poor stability. It is beneficial to improve the consistency and quality of conchospore release and provides a new technical approach for the production of *Porphyra yezoensis* seedlings. Attached Figure Description

[0017] Figure 1 Image of the strain under an electron microscope;

[0018] Figure 2 Bacterial growth curve;

[0019] Figure 3 : Response surface to optimal growth conditions;

[0020] Figure 4 : A diagram of strain phylogenetic relationships based on average nucleotide identity (ANI);

[0021] Figure 5 : Observation diagram of expansion rate;

[0022] Figure 6 : Observation diagram of the proportion of sporangial branches. Detailed Implementation

[0023] The present invention will now be described in detail with reference to the embodiments and accompanying drawings.

[0024] Example 1: Isolation of bacterial strains

[0025] Aqueous phase and filaments were extracted separately from the shell filaments of *Porphyra yezoensis* (GS) after 4 weeks of swelling, followed by serial dilutions, and the supernatant was collected for later use. The diluted bacterial suspension was spread onto 2216E solid medium and incubated at 26°C for 72 h. Single colonies with different morphologies were picked and streaked onto 2216E solid medium for purification. The purified strains were stored at -80°C with 20% (v / v) glycerol for later use.

[0026] Free filaments (ZD-1) were cultured in an incubator at 20 ± 0.5℃, with a light intensity of 1300 ~ 2500 Lx and a light-dark cycle of 12 L:12 D. The nutrient solution was Ningda III medium (nutrient solution composition: 990 nmol / L KNO3, 9 nmol / L FeSO4·7H2O, 57 nmol / L K2HPO4, 1 nmol / L MnSO4·H2O, 54 nmol / L Na2EDTA, 0.5 mg / L VB). 125 mg / L VB1, added at a ratio of 1:1000 mL (v / v) of nutrient solution to sterilized seawater.

[0027] 1. Morphological observation

[0028] Fresh cultures of the bacterial strain were observed under an optical microscope to examine the morphology, size, motility, and spore production. Under an electron microscope, (…) Figure 1 It is rod-shaped and is a Gram-negative bacterium.

[0029] 2. Growth curve

[0030] An appropriate amount of the bacterial strain was inoculated into 2216E liquid medium for activation and cultured continuously at 30 ℃ and 200 r / min for 24 h to ensure that the strain reached the logarithmic growth phase. The activated strain was then inoculated at a rate of 1% into 35 ml of 2216E liquid medium and cultured continuously at 30 ℃ and 200 r / min for 32 h. The absorbance (OD) of the culture medium was measured every 4 hours using a microplate reader. 600 ), and record the corresponding data. Origin was used to analyze the experimentally measured OD. 600 Data analysis was performed to obtain bacterial growth curves. The OD values ​​measured in the experiment were then analyzed to determine the bacterial growth curves. 600 The values ​​were analyzed, and the growth curve was fitted using the logistic equation to obtain the bacterial growth curve. The results are as follows: Figure 2 As shown: Bacteria grow slowly in the early stage of culture (around 0-8 hours); then (around 8-24 hours) they enter the logarithmic growth phase and multiply rapidly; after about 24 hours they enter the stationary phase.

[0031] 3. Optimal growth conditions of the strain

[0032] The optimal conditions for bacterial culture (temperature, pH, and N / P ratio of inorganic salts) were optimized using response surface methodology (RSM). The influencing factors and their levels are shown in Table 1. The pH was adjusted in 2216E liquid medium using NaOH (1 mol / L) and HCl (0.1 mol / L). After 8 h of culture (inoculum size 2%), the absorbance (OD) of the culture medium was measured using a microplate reader. 600 ).

[0033] Table 1: Influencing Factors and Levels

[0034]

[0035] The culture conditions data for each group are as follows: Figure 3 As shown, the optimal nitrogen-to-phosphorus ratio (N / P) for the strain is 1:5, the optimal pH is 8.2, and the optimal temperature is 30℃.

[0036] Example 2: Determination of the physicochemical properties of the strain

[0037] 1. Antimicrobial susceptibility testing of bacterial strains

[0038] The disk diffusion method was used. 100 μL of MT bacterial suspension, activated for 24 h at 30℃ and 200 r / min, was pipetted, diluted 10-fold with sterile seawater, and spread onto 2216E solid plates. In a clean bench, sterile forceps were used to gently place disks containing various antibiotics onto the plate. The plates were incubated upside down at 30℃ for 48 h. The diameter of the inhibition zones was observed and recorded. Based on the size of the inhibition zones and the instructions for use of the antibiotic disks, the susceptibility or degree of susceptibility of the strain to these antibiotics was determined.

[0039] The following antibiotics are listed: Penicillin G, Oxyphenidyl (oxazolidin), Ampicillin (ampicillin), Carbenicillin (carbenicillin), Piperacillin (piperazine penicillin), Cephalexin (cephalosporin IV), Cefazolin (cephalosporin V), Cephalexin (cephalosporin VI), Cefuroxime (Cefuroxime), Ceftazidime (Cefatacillin), Ceftriaxone (Ceftriaxone), Cefoperazone (Cefoperazone), Amikacin (Amikacin), Gentamicin, Kanamycin, Neomycin, Erythromycin, Ofloxacin (Ofos), Vancomycin, Polymyxin B, Compound Sulfamethoxazole, Furazolidone, Chloramphenicol, Clindamycin (Clindamycin).

[0040] Antimicrobial susceptibility testing showed that this strain was not sensitive to penicillin G, cephalexin, cefadroxil, cefuroxime, ceftazidime, polymyxin B, and trimethoprim-sulfamethoxazole; moderately sensitive to oxacillin, ampicillin, carbenicillin, cefazolin, tetracycline, and minocycline; highly sensitive to ceftriaxone, gentamicin, kanamycin, neomycin, erythromycin, norfloxacin, and vancomycin; and extremely sensitive to piperacillin, cefoperazone, amikacin, doxycycline, midecamycin, ofloxacin, ciprofloxacin, furazolidone, chloramphenicol, and clindamycin. This indicates that the bacterium has high sensitivity to most penicillins and most aminoglycosides. (General criteria are: inhibition zone diameter >20 mm indicates extremely sensitive, >15–20 mm indicates highly sensitive, 10–15 mm indicates moderately sensitive, and <10 mm indicates resistant). Physiological and biochemical detection of API 20NE was also performed.

[0041] Use a pipette to take 100 μL of the MT bacterial solution activated for 24 h under the culture conditions of 30°C and 200 r / min, add sterilized seawater to dilute it 10 times, then spread it on 2216E solid medium, perform inverted culture in a 30°C constant temperature incubator for 48 h, pick a single colony and place it in normal saline to prepare a bacterial suspension with a standard turbidity of 0.5 McFarland unit. Put the test strip into a culture box added with about 5 ml of water, add 110 μL of the bacterial suspension to the wells from NO3 to PNPG, and add liquid paraffin to GLU, ADH and URE until a protruding crescent shape is formed. Take 200 μL of the bacterial suspension and add it into the AUX medium, mix well and then add it to the wells from GLU to PAC, making sure that the liquid level must be flat or slightly convex. After marking, place the culture box in a 30°C constant temperature incubator for 24 h of culture, and after 24 h, interpret the test results in combination with the instruction manual.

[0042] The test results of API20NE are shown in Table 2. Nitrate reduction, arginine dihydrolase, esculin hydrolysis, gelatin hydrolysis, glucose assimilation, arabinose assimilation, mannose assimilation, mannitol assimilation, N-acetylglucosamine assimilation, maltose assimilation, potassium gluconate assimilation, adipic acid assimilation, malic acid assimilation, sodium citrate assimilation, and cytochrome oxidase show positive reactions; nitrite reduction, indole production, glucose fermentation, urease, β-galactosidase, capric acid assimilation, and phenylacetic acid assimilation show negative reactions.

[0043] Table 2: Bacterial API20NE test result table

[0044] Description Outcome Assimilation Outcome Nitrate reduction ++ Glucose ++ Nitrite reduction - Arabinose + Indole - Mannose + Glucose fermentation - Mannitol + Arginine dihydrolase ++ Acetylglucosamine ++ Urease - Maltose ++ Esculin ferric citrate ++ Potassium gluconate ++ Gel hydrolysis ++ Sheep wax acid - β-Galactosidase - Adipic acid + Cytochrome oxidase + Malic acid ++ Sodium citrate + Phenylacetic acid -

[0045] Note: "+" means positive; "-" means negative.

[0046] 2. 16S rDNA gene sequence analysis of the strain

[0047] Bacterial DNA was rapidly frozen in liquid nitrogen, and genomic DNA was extracted using the TIANamp Bacterial DNA Extraction Kit (DP302). Universal primers 27F (5'-AGA GTT TGA TCC TGG CTC AG-3') and 1492R (5'-TAC GGC TAC CTT GTT ACG ACT T-3') were selected for amplifying the bacterial 16S rRNA gene. The PCR program consisted of 35 cycles of 96°C pre-denaturation for 5 minutes, followed by 20 seconds of denaturation at 96°C, 20 seconds of annealing at 62°C, 30 seconds of extension at 72°C, and a final extension at 72°C for 10 minutes. PCR products were detected by 1.0% agarose gel electrophoresis. The primers used for gene sequencing were the same as those used for amplification. Sequencing results were analyzed using BLAST sequence similarity comparison in the NCBI database. A 16S rDNA phylogenetic tree was constructed using MEGA11.0 with neighbor-joining (repetition F1000).

[0048] The full-length 16S rRNA gene sequence of the bacteria is 1381 bp. Homology analysis of the 16S rRNA gene sequence using EzBioCloud showed that the MT strain and Roseivirga spongicola strain UST030701-084 shared a sequence similarity of 99.78%. Subsequent genome sequencing and ANI analysis identified it as Roseivirga spongicola UST030701-084. Figure 4 This strain is currently deposited at the China Center for Type Culture Collection (Wuhan), with accession number CCTCC M 20242610.

[0049] Example 3: The promoting effect of the strain on Porphyra yezoensis

[0050] 1. The effect of the strain on the expansion of filaments

[0051] An appropriate amount of bacterial strain was inoculated into 2216E liquid medium for activation and cultured continuously at 26℃ and 200 r / min for 24 h. The culture was then centrifuged, and the supernatant was collected. An appropriate amount of free filaments was placed in a 6-well plate containing 10 mL of algal culture medium. Bacteria were inoculated into the algal culture medium at a ratio of supernatant to sterile seawater of 1:500 (v / v). Normally cultured filaments served as a control, with three replicates per well. The culture conditions were: temperature 28℃, light intensity 500 Lux, photoperiod 8 L:12 D, nutrient salts NP (100 ppm:20 ppm), and salinity 28‰. Optical microscopy observations were performed at weeks 0, 1, 2, 3, 4, 5, 6, and 7, and the filament swelling ratio was calculated using Photoshop.

[0052] Compared to the control group, the Roseivirga sp. group showed a greater number of enlarging cells and a faster enlargement rate within the same timeframe. On day 14 of enlargement, the number of enlarging cells in the Roseivirga sp. group was four times that of the control group. Figure 5 ).

[0053] 2. Filament swelling mode

[0054] Select 1-2 healthy, free-growing filamentous algal spheres under normal growth conditions, break them up, and aliquot them into six-well plates. Incubate in the dark for 3 days. After 3 days, remove the seawater from the six-well plates and add 10 mL of sterilized seawater diluted with phosphorus (P) at a ratio of 1:1000. Add 20 μL (1:500) of SMT bacterial supernatant to the experimental group. Culture conditions are: temperature 28℃, light intensity 500 Lx, photoperiod 8 L:12 D. Change the water every 7 days and observe under an inverted microscope every two days.

[0055] The supernatant culture of strain Roseivirga sp. for 4 weeks affected the formation of sporangial branches as follows: Figure 6 As shown, compared with the control group, the proportion of sporangial branches formed by Roseivirga sp. after 4 weeks of treatment was significantly increased, with the swelling rate of the control group being 18.3% and that of the Roseivirga sp. group being 48.8%.

[0056] In summary, the strain and its metabolites of this invention have a strong promoting effect on the swelling of filaments and the formation of sporangial branches in Porphyra yezoensis. The application of this strain can provide a new and efficient microbial growth promoter for the artificial seedling cultivation of Porphyra yezoensis, which is expected to shorten the seedling cycle and improve sporulation efficiency, and has important application potential and economic value.

Claims

1. A pink rod-shaped bacterium ( Roseivirga sp. ), characterized in that, The preservation number of the pink rod-shaped bacteria is CCTCC NO: M 20242610.

2. The use of the pink rod-shaped fungus according to claim 1 in the preparation of a formulation for promoting the development of filamentous sporangial branches of *Porphyra yezoensis*.

3. The application as described in claim 2, characterized in that, The preparation is a microbial bacterial solution.

4. A microbial preparation, characterized in that, The microbial preparation contains live bacteria of the pink rod-shaped bacteria as described in claim 1.

5. The microbial preparation according to claim 4, characterized in that, The microbial preparation further comprises the fermentation supernatant of the pink rod-shaped bacteria as described in claim 1.

6. A method for promoting the maturation of filamentous sporangial branches of *Porphyra yezoensis*, characterized in that, The method involves culturing free filaments of *Porphyra yezoensis* by adding the pink rod-shaped bacteria and / or its fermentation supernatant as described in claim 1 to the filament culture medium.

7. The method as described in claim 6, characterized in that, The cultivation conditions were: temperature 28℃, light intensity 500Lx, and photoperiod 8L:16D.

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