Kelp endophyte prelisteria megaterium H403 and application of kelp endophyte prelisteria megaterium H403

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a kelp endophyte, i.e., presteria megaterium H403 and an application of the kelp endophyte, i.e., presteria megaterium H403. In order to solve the problems that the kelp seedling raising period is long and the cost is high, an endophytic bacterium is separated from kelp and is named as presteria megaterium H403, a co-culture experiment shows that the strain can remarkably improve the germination rate and the growth rate of kelp seedlings, and the length of kelp in a treatment group added with 14 * 10 < 6 > cfu bacterial liquid is 3.6 times that of a control group. The invention discloses a molecular mechanism for promoting the growth of the kelp through multiple ways by virtue of the Pristeria megaterium H403, a theoretical basis is provided for developing a microorganism-assisted seedling raising technology, the kelp culture cost is expected to be reduced, and the sustainable development of the industry is promoted.

Description

Technical Field

[0001] This invention belongs to the field of microbial and aquaculture technology, and particularly relates to a kelp endophyte, Priestia megaterium H403, and its applications. Background Technology

[0002] Algae cultivation, as an important component of aquaculture, plays a crucial role in maintaining the health of marine ecosystems and promoting the development of the blue economy. Kelp covers approximately one-quarter of the world's coastlines and is the second largest marine ecosystem after seagrass. The circulating cooling water seedling cultivation technology, adopted since the 1950s, has become the standard production process in the algae cultivation industry through the cultivation of "summer spores" and continuous optimization. While this technology solves the technical challenges of high-temperature seedling cultivation in summer, it still faces significant cost pressures: firstly, it requires strict control of the cultivation water temperature at around 12°C; secondly, it necessitates the deployment of professional personnel for daily monitoring and management of growth. The entire seedling cultivation cycle typically lasts more than three months, leading to a substantial increase in production costs. Specifically, costs consist of two main parts: electricity consumption, with statistics showing that electricity consumption during peak seedling cultivation season increases 80 times compared to the off-season, averaging 277,189 kWh per month; and labor management, with related research indicating that this expenditure accounts for 12.8% of the total seedling production cost. Therefore, shortening the seedling cultivation time can reduce cultivation costs and generate more profits.

[0003] In recent years, the application potential of microbial regulation technology in algae cultivation has received widespread attention. Currently, there are no mature cases of large-scale application of microbial technology for kelp seedling cultivation. Preliminary experiments show that physical cell wall disruption can reintroduce *Bacillus subtilis* into *Ulvasp.*, significantly altering the diversity and abundance of the algal microbiome. These findings provide important references for the future development of microbial-assisted seedling cultivation technologies. Theoretical studies suggest that specific strains may promote seaweed growth by secreting plant hormone analogs or activating antioxidant systems. Large algae are more sensitive to microbial colonization during their early growth stages (seedling period), and some large algae can even transfer the colonized beneficial microorganisms.

[0004] As research into microbial-host interactions deepens, increasing evidence suggests that endophytic bacteria and algae can form a close, mutually beneficial symbiotic relationship, significantly promoting host growth, development, and stress resistance through various mechanisms. Drawing on established paradigms of plant growth-promoting (PGP) mechanisms in terrestrial plants, endophytic bacteria typically promote host growth through nitrogen fixation, plant hormone synthesis (such as IAA), biocontrol (such as secretion of antimicrobial substances), siderophore production, and enhanced mineral solubility. These mechanisms also hold significant reference value in algae-microbe interactions, particularly in the cultivation of large, economically important algae such as kelp. Analyzing the PGP functions of endophytic bacteria can provide a theoretical basis for developing aquaculture-specific microbial preparations, thereby optimizing kelp seedling cultivation processes and achieving greener, more efficient aquaculture. This research approach not only expands the application of terrestrial plant PGP mechanisms in aquaculture but also provides a new research direction for algae-microbiome engineering. Summary of the Invention

[0005] To address the problems of long seedling cultivation cycles and high costs in the existing kelp technology, this invention provides a kelp endophyte, *Priestia megaterium* H403, and its applications.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: This invention isolates an endophytic bacterium from kelp. This strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on August 22, 2025, with accession number CGMCC No. 35552. It is named *Priestia megaterium* H403.

[0007] This invention identified key gene clusters in strain H403 related to IAA synthesis, siderophore production, phosphate dissolution, and nitrogen fixation through whole-genome sequencing, and preliminarily elucidated the mechanism by which H403 promotes the growth of kelp seedlings. These findings confirm the application value of strain H403 in kelp seedling cultivation and provide new technical ideas for large-scale kelp production. This research lays an important foundation for subsequent research and practical applications of microbial-assisted kelp cultivation.

[0008] The genome sequence of the kelp endophyte H403 includes multiple functional gene clusters related to plant vitality, mainly including: phosphate transport system gene cluster, nitrogen fixation related gene cluster, siderophore biosynthesis gene cluster, and indole-3-acetic acid (IAA) synthesis pathway related gene cluster.

[0009] The endophytic bacterium *Priestia megaterium* H403 described above is adapted to the kelp seedling environment (10-25°C, 1-5% salinity) and does not have the ability to degrade alginate.

[0010] The present invention also discloses the application of the kelp endophyte *Priestia megaterium* H403 in seaweed cultivation.

[0011] Preferably, the seaweed is kelp.

[0012] Preferably, the endophytic bacterium *Priestia megaterium* H403 of kelp is used in kelp seedling cultivation.

[0013] A method for cultivating kelp seedlings, utilizing any of the above-mentioned kelp endophytic bacteria, *Plastrum megaterium*. Priestia megaterium The H403 strain is used for kelp seedling cultivation. The specific cultivation method includes: storing the H403 strain at -80℃; immersing an inoculation loop in the bacterial solution and streaking it onto a freshly prepared LB solid medium plate until single colonies grow; selecting H403 colonies using an inoculation loop and then inoculating them into liquid LB medium; centrifuging the resulting liquid and washing it twice with sterile seawater to obtain the H403 strain; adding the H403 strain to seawater for kelp seedling cultivation.

[0014] This invention addresses the problems of long seedling cultivation cycles and high costs associated with kelp. An endophytic bacterium strain was isolated from kelp and named *Priestia megaterium* H403. Co-culture experiments revealed that this strain significantly improved the germination rate and growth rate of kelp seedlings, especially with the addition of 14 × 10⁻⁶ ppm. 6 The kelp length in the CFU bacterial solution treatment group was 3.6 times that of the control group. Whole-genome sequencing analysis showed that the H403 genome contains 5,458 coding sequences, including plant growth promotion (PGP) related genes such as nitrogen fixation (nifU gene), phosphate solubilization (pst system), siderophore synthesis (schizokinen gene cluster), and indole-3-acetic acid (IAA) synthesis (tryptophan pathway gene). In vitro experiments confirmed that H403 can secrete siderophores, solubilize organic phosphorus, and produce 14.98 mg / L of IAA under tryptophan induction. In addition, H403 adapts to the kelp seedling environment (10-25°C, 1-5% salinity) and does not produce alginate lyase, thus avoiding damage to the kelp's defense barrier. This invention reveals the molecular mechanism by which H403 promotes kelp growth through multiple pathways, providing a theoretical basis for the development of microbial-assisted seedling technology, which is expected to reduce kelp farming costs and promote the sustainable development of the industry. Attached Figure Description

[0015] Figure 1 Figure showing the results of strain identification and growth characteristics experiments; Figure 2 Figure 1 shows the growth results of the bacterial strain under abiotic stress. A. Growth of H4O3 at different salinities for 24 hours. B. Growth curve of H4O3 at 10℃. C. Growth of H4O3 after 12 hours (blue) and 72 hours (red) at different temperatures. Figure 3 Figure 1 shows the experimental results of H403's plant growth promoting activity: (A) Organic phosphate detection; (B) Potassium solubilization capacity detection; (C) Siderophoresis detection; (D) Indole-3-acetic acid (IAA) detection; (E) Alginate enzyme detection; (F) Nitrogen fixation detection. Figure 4 Figure 1 shows the experimental results of kelp co-cultured with different concentrations of H403 bacteria. (A) Germination rate; (B) Length; (C) Width; (D) SRG determination. Asterisks indicate significant differences between treatments (*p<0.05, **p<0.01, ***p<0.001 Wilcoxon test). Figure 5 A:H403 Genome Map: The outermost circle represents the genome size; the second and third circles represent genes on the positive and negative strands of the genome, respectively; different colors represent different COG functional classifications; the fourth circle is a triangle representing tRNA (green for the positive strand and blue for the negative strand), and a square representing rRNA (red for the positive strand and purple for the negative strand); the fifth circle is the genomic GC content; the sixth circle is the genomic GC offset. AZ show the functional classifications of CDS genes in the chromosomes. B: Heatmap of average nucleotide identity (ANI) between strain H4 and eight closely related phylogenetic species; Figure 6 Metabolic pathways of tryptophan synthesis (red), nitrogen metabolism (blue), betaine and proline synthesis (green) in strain H4, as well as proteins related to Na+ / H+ reverse transport (yellow). Detailed Implementation

[0016] The present invention will now be described in detail. Before proceeding with the description, it should be understood that the terminology used in this specification and the appended claims should not be construed as limited to its general or dictionary meaning, but rather should be interpreted according to the meaning and concept corresponding to the technical aspects of the invention, based on the principle that the inventors are allowed to appropriately define the terms for the best interpretation. Therefore, the description presented herein is merely a preferred example for illustrative purposes and is not intended to limit the scope of the invention. It should be understood that other equivalents or modifications can be obtained from it without departing from the spirit and scope of the invention.

[0017] The following embodiments are merely examples illustrating implementations of the present invention and do not constitute any limitation on the present invention. Those skilled in the art will understand that modifications made without departing from the spirit and concept of the present invention fall within the protection scope of the present invention. Unless otherwise specified, the reagents and instruments used in the following embodiments are commercially available products.

[0018] Example 1: Isolation of Priestia megaterium H403 The farmed kelp was obtained from a farm in Xiapu County, Ningde City, Fujian Province.

[0019] The specific isolation method is as follows: After rinsing the kelp sample with sterile water to clean the surface, take a 5cm*5cm kelp leaf and transfer it to a laminar flow hood. Surface sterilize it by repeatedly rinsing with 75% ethanol (1min), 3% sodium hypochlorite solution (2min), and sterile water (4-5 times). Transfer it to a sterile mortar and crush it. Add 1ml of sterile water to prepare a bacterial suspension. Take 200μL and spread it evenly on LB solid medium. Verify surface sterility by spreading it with the final rinsing solution. After incubating in a 37℃ constant temperature incubator for 24h, observe the colony morphology. Purify the dominant strain by continuous streak isolation method, and finally obtain a kelp endophyte, Priestia megaterium H403.

[0020] Example 2 Identification of Priestia megaterium H403 The strain *Priestia megaterium* H403 isolated in Example 1 was identified as follows: First, colony morphology (color, shape, etc.) was observed using an optical microscope (Nikon Ti-E-A1R, Tokyo, Japan), and typical single colonies were selected for further analysis. Phenotypic identification was performed according to Bergey's Handbook of Systematic Bacteriology.

[0021] Subsequently, the strain was directly sent to Sangon Biotech (Qingdao) for 16S rDNA sequencing using universal primers 27F / 1492R, and compared with the NCBI database. Based on the sequence data, a phylogenetic tree was constructed using the maximum likelihood (ML) method with MEGA 11.0 software and a bootstrap value of 1000 replicates.

[0022] Morphological observation showed that single colonies of *Priestia megaterium* H403 on LB agar were milky white, opaque, round cells, raised with a smooth, moist surface and neat, slightly translucent edges. Microscopic evaluation also showed typical characteristics of Gram-positive bacteria, exhibiting a rod-like shape. A partial 16S rRNA gene sequence of H403 was amplified and compared with 16S rRNA gene sequences stored in the NCBI database. A phylogenetic tree was constructed using MEGA 11.0, incorporating the BLAST results of *Priestia megaterium* H403, and the results are shown below.

[0023] Mean nucleotide identity (ANI) is an indicator of the relationship between two genomes at the nucleotide level. ANI is defined as the average base similarity between homologous segments of two microbial genomes. It is characterized by a high degree of differentiation between closely related species; generally, an ANI ≥ 95% is considered to indicate the same species. Based on 16S rRNA gene and ANI analysis, H403 and *Primatex megaterium* were compared. P. megaterium Closely related. H403 and Priestia megaterium NBCR 15038 clustered on the same branch. H403 was preliminarily identified as *Primatellis megacephala*. P. megaterium.

[0024] Example 3: Genome sequencing and functional annotation of Priestia megaterium H403 (1) Genome sequencing of Priestia megaterium H403 A single colony of *Priestia megaterium* H403 from the culture plate was inoculated into 20 ml of liquid LB medium and incubated at 28°C with shaking at 180 rpm for 12 hours. 1 mL of the bacterial culture was then mixed with 50% glycerol and transferred to a centrifuge tube.

[0025] Priestia megaterium H403 samples were sent to Yunsitop Biotechnology Co., Ltd. (Tianjin, China) for genome assembly using a combination of Nanopore and Illumina sequencing. The ONPV5 assembly workflow was employed, including sample quality control, library construction, library quality control, and library sequencing. PacBio sequencing technology used SMRT chips as the sequencing vector. DNA polymerase bound to a template in the nanopores of the SMRT chip, and the dNTP mixture was labeled with four-color fluorescent dyes. During the base pairing stage, different bases were labeled with different wavelengths and peak values ​​based on the light intensity. SMRT Link version 8.0 software was used to process external PB data to obtain circularly shared sequencing (CCS) data. After further filtering of short reads (<2,000 bp in length), the total dataset was obtained. The filtered CCS data was assembled using Hifiasm v0.12 (r304) software, and Pilon version 1.22 software was used for second-generation data correction to obtain a more accurate genome for subsequent analysis. Average nucleotide identity (ANI) analysis was performed. The ANI value of H403 compared to eight closely related strains was calculated using the JspeciesWS online tool, and the results are presented as a heatmap.

[0026] (2) Genomic composition analysis and functional annotation Genomic composition analysis included coding gene prediction, repetitive sequence prediction, CRISPR (Clustered Regularly Interspaced Palindromic Repeats) sequence prediction, pseudogene prediction, gene island prediction, prophage prediction, gene cluster prediction, promoter prediction, and paralogous gene prediction. Biosynthetic gene clusters (BGCs) in bacterial genome sequences were identified and analyzed using antiSMASH v5.0.0 software, and the results were combined with NCBI BLAST alignment analysis. Sequencing results were compared with seven databases—Non-Redundant Protein Database (Nr), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), COG, Pfam, TIGRfam, and uniprot—for gene function annotation and comparison with annotation information from each database. Using the predicted genome data, Circos v0.66 software was used. From the KEGG database, genes related to indole-3-acetic acid (IAA) production, phosphate solubility, and nitrogen fixation were analyzed. Gene clusters from H403 secondary metabolites were investigated.

[0027] (3) Predicted gene clusters involved in the synthesis of secondary metabolites in H403 To identify the gene clusters involved in secondary metabolite synthesis in strain H403, antiSMASH software prediction and NCBI BLAST comparative analysis were performed. The results showed that strain H403 encodes seven gene clusters involved in secondary metabolite synthesis. Among them, one type III polyketide synthase, three terpenes, one terpene precursor, one phosphonate, and one siderophore cluster (Table 2) showed 63% similarity to known gene clusters encoded by carotenoids. Additionally, the similarity to the gene cluster encoded by schizotycin was 66%. Furthermore, this genome also encodes five gene clusters presumed to be responsible for the biosynthesis of unknown metabolites, with initial functional annotations as follows: type III polyketide synthase, terpene synthase, phosphonate biosynthesis gene, terpene precursor modifying enzyme, and siderophore nonribosomal peptide synthase. These require further investigation.

[0028] (4) Genes effectively linked to PGP in the H403 genome The PGP properties of H403, including phosphate dissolution, nitrogen fixation, siderophore production, IAA production, and ammonia production, have been confirmed in vitro. We screened genes in the H403 genome associated with PGP activity.

[0029] In the phosphate metabolism pathway ( Figure 2 This is consistent with our previous phosphate dissolution experiments. H403 can dissolve organic phosphates, suggesting that phosphate transport proteins in the H403 genome may be closely related to phosphate dissolution (Table 3). Whole-genome sequencing analysis revealed that H403 carries a series of genes related to IAA synthesis, including the complete tryptophan biosynthesis gene cluster trpABCDEFS (Table 3), which are closely related to the synthesis of tryptophan, the precursor of IAA. Although metabolic pathway annotation based on the KEGG database failed to reconstruct the complete IAA synthesis pathway, we successfully identified two key IAA synthesis nodes: first, indole-3-acetaldehyde can be converted into IAA by the catalysis of aldehyde dehydrogenase (ALDH); second, (Indol-3-yl)acetamide can generate IAA under the action of amidase (E3.5.1.4) (Supplementary Figure). Notably, although genomic analysis did not fully elucidate the IAA synthesis mechanism, in vitro fermentation experiments clearly confirmed that strain H403 has a significant IAA synthesis capacity, with a yield of 14.98 ± 0.23 mg / L.

[0030] We also identified a group of genes associated with nitrogen fixation in H403 (Table 3). This demonstrates H403's ability to produce nitrogen. Two ammonia-producing pathways were identified in the nitrogen metabolism pathway map of H403. The first includes enzymes encoded by gudB and rocG, which catalyze the reduction of L-glutamate to ammonia, and the other includes enzymes encoded by nasD and nasE, which catalyze the reduction of nitrite to ammonia. fhuD, fhuG, fhuB, and fhuA were annotated as encoding iron-chelating transport proteins, which may be related to siderophores (Table 3). Furthermore, the siderophore gene cluster was located on chromosomes (3961685-3978260) as predicted by antiSMASH software, and the positive results on CAS medium further indicate that H403 is capable of producing siderophores.

[0031] This invention, through genomic analysis, predicts that strain H403 contains seven gene clusters for secondary metabolite synthesis, including a gene cluster that perfectly matches the synthesis of known schizokinin siderophores (63% similarity) and carotenoids (66% similarity), as well as several gene clusters of terpenes, phosphonates, and type III polyketide synthases with unknown functions. These secondary metabolites may promote kelp growth through multiple mechanisms: previous studies have shown that appropriate iron supplementation has a significant promoting effect on kelp seedling cultivation. Specifically, iron ions can effectively stimulate the growth and development of female gametophytes in kelp. In the prediction of the whole genome of strain H403, a gene cluster of siderophores was found, which can enhance the efficiency of iron acquisition, thereby correspondingly increasing the iron content during kelp seedling cultivation. Carotenoids may provide antioxidant protection, while other metabolites may participate in antimicrobial defense or phosphorus metabolism regulation. In particular, the expression of the schizokinin siderophore gene cluster is highly consistent with the in vitro CAS detection results, confirming the competitive advantage of this strain in iron-limited environments.

[0032] Example 4: Non-biological in vitro stress experiment of Priestia megaterium H403 strain The growth of H403 at different temperatures (5–25°C) and salt concentrations (1–7%) was examined to determine its ability to withstand two abiotic stresses. Absorbance at 600 nm was measured using a spectrophotometer. These physiological tests were performed in triplicate.

[0033] 1. Experimental Method (1) Temperature growth experiment The isolated H403 was inoculated into LB liquid medium at a 1% inoculum and shaken at 180 rpm at 15, 20, and 25 °C. At 12 and 72 hours, 10 mL samples of the culture medium were taken, and the OD was measured. 600Value. Bacterial growth (OD) was measured every 6 hours at 5 and 10°C. 600 Then, a growth curve of the bacteria can be plotted.

[0034] (2) Salinity growth experiment The salinity of LB broth was adjusted to 1%, 3%, 5%, and 7% using NaCl. Bacteria were then inoculated into LB media at different salinity levels at a 1% inoculum and incubated for 24 hours at 28°C and 180 rpm in a shaker. The OD was determined... 600 The value is used to calculate growth.

[0035] (3) Experiments to determine PGP-like activity and alginate lyase activity We used the standard method for preparing antibiotic susceptibility testing tablets. Strain H403 was inoculated at a 1% inoculum on LB medium and cultured at 28°C and 180 rpm for 18 h with shaking. 200 μL of the bacterial suspension was then evenly added to a sterile antibiotic susceptibility testing tablet and dried at 28°C for 12 h to prepare the bacterial susceptibility testing tablet.

[0036] (4) Determination of alginate lyase activity and potassium solubilization capacity Antimicrobial susceptibility testing plates containing H403 were inoculated onto screening plates using sodium alginate as the sole carbon source. The plates were then incubated at 28°C for 48 h. The ability of H403 to produce this hydrolytic enzyme was determined by observing whether a clear zone formed around the colony.

[0037] (5) Nitrogen fixation and phosphate dissolution of H4O3 Modified agar media were prepared, including an inorganic phosphate medium containing 2% (w / v) tricalcium phosphate (Ca3(PO4)2) and an organic phosphate medium using lecithin as the phosphorus source. H4O3-containing antimicrobial susceptibility assay plates were then inoculated onto these plates and incubated at 28°C for 7 days. After incubation, the formation of a transparent phosphate-solubilizing zone around the colonies was observed. The presence or absence of this zone assessed the phosphate-solubilizing ability of H4O3. Nitrogen fixation capacity was measured using Ashby nitrogen-free medium (mannitol: 10 g, KH2PO4: 0.2 g, MgSO4⋅7H2O: 0.2 g, NaCl: 0.2 g, CaSO4⋅2H2O: 0.1 g, CaCO3: 5 g, agar: 15 g, distilled water: 1,000 ml). H4O3 was inoculated into Ashby medium at 28°C for 7 days to determine its activity.

[0038] (6) Detection of ferrocarriers in H403 To confirm the expression of the siderophore gene cluster, we inoculated PGPB isolates onto chromazurol S (CAS) agar plates (CAS: 0.0605 g, Na2HPO4·12H2O: 1.2135 g, NH4Cl: 0.125 g, KH2PO4: 0.0375 g, Hexadecyl trimethyl ammonium bromide (CTAB): 0.0729 g, FeCl3·6H2O: 0.002645 g, NaH2PO4·2H2O: 0.29525 g, NaCl: 0.0625 g). The plates were incubated at 28°C for 2–4 days. We observed whether an orange-red halo appeared around the colonies. The experiment was repeated three times, and the pore size was measured after 48 h to analyze the siderophore capacity.

[0039] (7) Determination of indole-3-acetic acid (IAA) production Based on the standard curve of IAA, the IAA content per unit volume of fermentation broth was calculated. The strain was cultured in LB medium for 18 h and then transferred at a 1% inoculum to LB medium containing 0.1% L-tryptophan. The strain was cultured at 28°C for 2 days, followed by centrifugation at 1×10⁻⁴ r / min for 5 min. A total of 1 ml of fermentation supernatant was mixed with 2 ml of Fe-H₂SO₄ solution (4.5 g·L⁻¹ FeCl₃⋅6H₂O, 10.8 mol·L⁻¹ H₂SO₄, diluted to 100 ml with distilled water) and incubated in the dark for 1 h. The IAA concentration was estimated by measuring the absorbance of the sample at 540 nm.

[0040] (8) Potassium solubilization experiment H403 was inoculated onto a selective solid culture medium containing potassium feldspar using the streak plate method. The culture was incubated at 28°C for 4 days. During the incubation period, the surface of the medium was observed daily. The appearance of colonies indicated that the test strain possessed potassium-solubilizing ability and could utilize potassium from potassium feldspar, thus proving its potassium-utilizing capacity.

[0041] 2. Experimental Results (1) Growth characteristics of H403 We tested the tolerance of H403 under abiotic stress conditions of temperature (5-25℃) and salinity (1-7%) by measuring its absorbance at 600 nm. The results showed that... Figure 2The optimal growth conditions for this strain are 20℃ and 1% salinity. Regarding temperature adaptability, H403 can grow in the range of 10-25℃, with a peak growth rate at 20℃. It can still maintain slow growth at 10℃, but growth is completely inhibited at 5℃. Regarding salinity tolerance, when the salt concentration exceeds 1%, the growth rate of the strain decreases significantly with increasing salinity, and it cannot grow at 7% salinity.

[0042] (2) Evaluation of PGP activity in strain H403 In vitro PGP activity assays of H403 showed that this strain can secrete siderophores, forming typical brown chelation zones on CAS culture media, indicating its highly efficient iron chelating ability; the formation of transparent oil droplet-like colonies on silicate medium proved its potassium-solubilizing activity. Further studies revealed that H403 exhibited specific solubilizing ability for organophosphate sources, forming obvious phosphate-solubilizing zones (5 mm in diameter) after 7 days of culture, but its solubilizing effect on inorganic phosphorus sources was not significant. We measured the nitrogen fixation of H403 in nitrogen-free medium. We found that H403 can grow under nitrogen-free conditions. Notably, quantitative detection using the Salkowski colorimetric method showed that H403 produced 14.98 ± 0.23 mg / L (n=3) of IAA under tryptophan-induced conditions, demonstrating its IAA synthesis ability. Furthermore, we identified alginate enzyme in H403, and the result was negative. This indicates that H403 itself cannot produce this enzyme and cannot degrade alginate.

[0043] (3) Analysis of H403's ability to grow in seawater environment We also analyzed the ability of H403 to grow in a seawater environment, one being Na... + / H + The expression of the retrotransport genes nhac and mnhABCDEFG enables the transport of Na+ from bacteria via proteins encoded by these genes. + They are excreted from the body. Another process involves synthesizing protective substances within the body: betaine and proline. The first enzyme, encoded by gbsB and betB, catalyzes choline into betaine; the other includes enzymes encoded by rocF and rocD, which catalyze the conversion of arginine into proline. Their ability to thrive in high salinity environments, ensuring their normal growth and development, indirectly demonstrates their capacity to grow under high salinity conditions.

[0044] The adaptability of strain H403 to various stress conditions indicates its potential to function effectively in indoor kelp seedling cultivation environments. Temperature adaptability experiments showed that the strain could grow within the range of 10-25℃, with peak growth rate at 20℃. Notably, it maintained stable growth activity even at 10℃, which closely matches the optimal temperature range for kelp seedling cultivation (around 12℃). Salinity tolerance tests showed that H403 exhibited good growth under salinity gradients of 1-5%, fully adapting to the seawater cultivation environment (natural seawater salinity approximately 3.5%). This dual adaptability physiologically confirms the feasibility of applying H403 in indoor kelp seedling cultivation systems.

[0045] Genomic analysis revealed that strain H403 contains two key genes, gbsB and betB, involved in the biosynthesis of glycine betaine, consistent with previous reports. Similar to the phenomenon observed in *P. megaterium*, proline accumulation in H403 also exhibited salt stress dependence, significantly increasing only under high-salt conditions. Studies have shown significant differences in the distribution of salt-stress-resistant genes among different salt-tolerant bacteria; for example, all five species of *Halophilus* are rich in gene clusters related to the synthesis of salt-tolerant substances. These salt-tolerant substances synthesized by bacteria (including betaine, proline, trehalose, glutamate, and tetrahydropyrimidine) are called compatibility substances, which maintain intracellular homeostasis by regulating osmotic pressure. In strain H403, proline and betaine were identified as the main salt stress-responsive compatibility substances.

[0046] In addition, this strain also uses Na + / H + The activity of antitransport proteins removes excess salt ions from the cell, and this synergistic mechanism enables the cell to maintain normal growth in high-salt environments. Notably, monosodium glutamate (MSG), as a key metabolic node, not only connects the carbon and nitrogen metabolic network but is also a direct precursor to proline biosynthesis. These findings strongly demonstrate the potential application of the endophytic bacterium H403 isolated from kelp in indoor kelp seedling cultivation.

[0047] Existing research indicates that marine microorganisms can promote algal growth and development through a variety of complex physiological and biochemical mechanisms. These include direct effects on algal metabolism, such as IAA biosynthesis, siderophore secretion, and nitrate reduction, as well as synergistic effects that indirectly regulate algal physiological states through biofilm formation, quorum sensing signal transduction, and reactive oxygen species scavenging systems.

[0048] Based on the whole-genome sequencing data of strain H403, we have thoroughly elucidated its potential direct growth-promoting mechanisms (e.g., nitrogen fixation, phosphorus solubilization). These functional characteristics are highly compatible with the growth requirements of kelp (Saccharina japonica), particularly in the acquisition and utilization of nitrogen (N) and phosphorus (P), the two most critical limiting nutrients in kelp cultivation. In traditional kelp farming, large amounts of nitrogen and phosphorus are typically supplemented in the form of chemical nutrients. The H403 strain discovered in this invention exhibits the potential to replace or supplement chemical fertilizers through microbial pathways. Genome analysis shows that this strain possesses nutrient acquisition capabilities similar to those reported in Priestia megaterium (formerly Bacillus megaterium), including nitrogen fixation gene clusters (NIFs), organophosphate solubilization systems (PSTs), and siderophore synthesis genes (such as the schizokinen gene cluster). The presence of these functional genes provides the molecular basis for its promotion of kelp growth.

[0049] Genome analysis of strain H403 revealed a relatively concise nitrogen-fixing genome, containing only one core gene, nifU. This gene encodes a protein that plays a crucial role in the assembly of the nitrogenase system, as previously demonstrated. The protein encoded by nifU is involved in the assembly and transport of [Fe-S] clusters. This differs from terrestrial plants, where nitrate is the primary form of dissolved inorganic nitrogen absorbed and fixed by organisms in seawater. Thus, strain H403 can convert nitrate in seawater into a form that kelp can absorb (bioactive ammonium). In the ocean, some organisms are also capable of this type of biological nitrogen fixation.

[0050] Genome analysis revealed that H403 contains a complete phosphate transport system gene cluster (pstSABC), and in vitro experiments confirmed its ability to dissolve organic phosphorus. Other endophytic bacteria, such as *Klebsiella variicola*, *Staphylococcus cohnii*, and *Rahnella aquatilis* HX2, have also been reported to dissolve phosphate. Notably, phosphorus, as the second most important macronutrient after nitrogen, is crucial for the metabolism, growth, and development of algae. Although different forms of phosphorus exist in seawater, most phosphorus is difficult for algae to absorb directly due to its tendency to form insoluble precipitates with metal ions or to compete with other organisms. The phosphorus-dissolving ability exhibited by strain H403 may be one of the important mechanisms by which it promotes the growth of its host algae.

[0051] The negative result in in vitro alginate testing further demonstrates that strain H403 can be used for kelp seedling cultivation. Kelp possesses a physical defense layer—alginate—that protects it from external threats. Many pathogenic microorganisms (such as alginate-degrading bacteria) destroy this protective structure by secreting alginate-lysing enzymes, thereby invading kelp tissue and causing disease. It is precisely because strain H403 lacks alginate-degrading capabilities that it can establish a symbiotic relationship with the host and promote kelp growth without disrupting the kelp's natural defense mechanisms.

[0052] Bacterial synthesis of indole-3-acetic acid (IAA) primarily occurs through four tryptophan-dependent metabolic pathways (Trp): the indole-3-pyruvate pathway (IPyA pathway), the tryptophan pathway (TAM pathway), the indole-3-acetonitrile pathway (IAN pathway), and the indole-3-acetamide pathway (IAM pathway). While there is no definitive evidence that the IPyA or TAM pathways in strain H403 are complete, IAA production experiments using tryptophan addition demonstrate that H403 can utilize tryptophan to produce IAA. Previous studies have shown that some bacteria can synthesize IAA through multiple incomplete pathways. Strain H403 contains four aldehyde dehydrogenases (ALDH) and one amidase (E3.5.1.4), which can convert indole-3-acetaldehyde and indole-3-acetamide into IAA, respectively. It is well known that L-trypsin is a key precursor and core regulator in IAA biosynthesis, and bacterial IAA synthesis is currently predominantly tryptophan-dependent. Through genomic analysis of strain H403, we also discovered that strain H403 possesses a complete tryptophan biosynthesis pathway. As a key growth hormone, IAA (indole-3-acetic acid) directly promotes plant growth and development by regulating cell division and elongation. Studies have shown that the biosynthesis, transport, and signal transduction pathways of IAA jointly mediate this physiological effect.

[0053] Example 5: Kelp Co-culture Experiment 1. Experimental Methods The *Priestia megaterium* H403 strain was stored at -80°C. An inoculation loop was immersed in the bacterial solution and streaked onto freshly prepared LB agar plates at 28°C until single colonies appeared. H403 colonies were selected using an inoculation loop and then inoculated into 100 ml of liquid LB medium at 28°C for 18 hours. The resulting liquid was then centrifuged at 10,000 rpm for 5 minutes and washed twice with sterile seawater. In this study, native Chinese kelp C12 was used. The bacterial strain was prepared at a concentration of 7 × 10⁻⁶ C / L of seawater. 6 14×10 6CFU was added, and a negative control was set up (30 ml of washing solution (sterile seawater) was added). Kelp was grown in a workshop (10 ± 2°C, 16 hours light and 8 hours dark, 2000 lux). To examine the effect of H403 on kelp growth and germination, germination rate, length, and width were measured twice a week after kelp began germination. Specific growth rate (SGR) was calculated using the formula: SGR(increase · d⁻¹) = (lnw₂ - lnw₁) / (T₂ - T₁), where w₁ = leaf length, width, or germination rate at time T₁, and w₂ = leaf length, width, or germination rate at time T₂. The experiment was repeated three times to obtain sufficient data.

[0054] 2. Effects of Priestia megaterium H4O3 on the growth of kelp seedlings We compared the culture group (7×10) under the same production conditions with the group that received the inoculum (7×10). 6 14×10 6 Compared with the control group, the overall effect of CFU (cfu) was 14×10 6 The germination rate of CFU with added H403 was the highest among the three groups, at 7×10⁻⁶. 6 Compared to the control group, the amount of CFU H403 added was also increased, and at day 23, it still had the highest germination rate among the three groups. We found that the addition of strain H403 had a significant impact on the growth of kelp. At day 27, the addition of 14×10⁻⁶ CFU H403 significantly affected the growth of kelp. 6 The kelp length of the CFU H403-treated group reached 1.6 times that of the control group, and by day 50, the two groups with different concentrations of the added kelp reached 3.6 times that of the control group. However, the width of the added kelp groups was not as significant as that of the control group. By calculating the growth (germination) rate after two months of indoor culture, we found a significant difference in length, reaching 1.5 times that of the control group, while the germination rate and width were not significantly different from the control. Finally, the seedlings reached the point where they could emerge from the sea one week earlier than the control group.

[0055] Normally, kelp seedlings can be harvested when they grow to about 1 cm and the water temperature is suitable. Preliminary experiments show that introducing strain H403 for co-cultivation can shorten the harvest time by about 7 days. This confirms that strain H403 can effectively shorten the indoor kelp seedling cultivation cycle and significantly reduce energy costs. This discovery provides important evidence for optimizing kelp seedling cultivation technology. Currently, we have preliminarily verified the application potential of this technology in kelp farming. The functional genes of strain H403 enable it to survive and promote growth in the kelp farming environment. This discovery provides the possibility for developing environmentally friendly microbial agents, which are expected to replace some chemical fertilizers in the future, promoting the development of algae farming towards a more environmentally friendly, economical, and sustainable direction.

[0056] Table 1. Genomic characteristics of H403 feature quantity Genome size (bp) 5448950 GC content (%) 38.05 Topology Ring Chromosome size (bp) 5174224 Number of chromosomes 1 Plasmid count 3 tRNA quantity 141 rRNA (5S, 16S, 23S) quantity 47 Number of protein-coding genes (CDS) 5458 Number of genome islands 1 CRISPR quantity 0 Number of gene clusters 7 Number of genes annotated into the NR database 5419 Number of genes annotated into the GO database 3624 Number of genes annotated into the KEGG database 2853 Number of genes annotated into the COG database 4813 Number of genes annotated into the Pfam database 4545 Number of genes annotated into the TIGRfam database 3070 Number of genes annotated into the UniProt database 5370 Table 2. Information on the isolated endophytic bacterium H403 from macroalgae. start site Termination site type Predicted product Similarity References 699,586 720,434 Terpenes Carotenoids 63% Halobacillus halophilus DSM 2266(1) 848,351 865,772 Phosphates - 1,539,949 1,581,034 Type III polyketide synthase - 2,260,952 2,282,820 Terpenes - 3,781,037 3,801,855 Terpenes - 3,952,685 3,987,260 Nonribosomal peptide-ferrocarrier Schizotypalin 66% Leptolyngbya sp. PCC 7376 (2) 4,360,261 4,381,157 Terpenoid precursors - Table 3. Predicted genes in the genome of kelp endophyte H403 associated with plant growth-promoting traits The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. A type of endophytic fungus in kelp, *Priscilla megaterium* Priestia megaterium H403, characterized in that, This strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on August 8, 2025, with accession number CGMCC No. 35552.

2. The endophytic fungus *Priscilla megaterium* as described in claim 1 Priestia megaterium H403, characterized in that, The endophytic fungus *Pleistocene megaterium* of kelp Priestia megaterium H403 was isolated from kelp tissue.

3. The endophytic fungus *Priscilla megaterium* as described in claim 1 Priestia megaterium H403, characterized in that, The endophytic fungus *Pleistocene megaterium* of kelp Priestia megaterium The genome sequence of H403 includes multiple functional gene clusters related to plant vitality, including: phosphate transport system gene cluster, nitrogen fixation-related gene cluster, siderophore biosynthesis gene cluster, and indole-3-acetic acid synthesis pathway-related gene cluster.

4. The endophytic fungus *Pristina megaterium* as described in claim 1. Priestia megaterium H403, characterized in that, The endophytic fungus *Pleistocene megaterium* of kelp Priestia megaterium H403 can secrete siderophores, dissolve organic phosphorus, and produce indole-3-acetic acid under tryptophan induction.

5. The endophytic fungus *Priscilla megaterium* as described in claim 1 Priestia megaterium H403, characterized in that, The endophytic fungus *Pleistocene megaterium* of kelp Priestia megaterium H403 is adapted to environments with temperatures of 10-25°C and salinity of 1-5%, and does not have the ability to degrade alginate.

6. The endophytic fungus *Pristinae megaterium* as described in claim 1. Priestia megaterium Application of H403 in seaweed cultivation.

7. The application according to claim 6, characterized in that, The seaweed in question is kelp.

8. The application according to claim 7, characterized in that, The endophytic fungus *Pristina megaterium* mentioned above Priestia megaterium H403 is used in kelp seedling cultivation.

9. A method for cultivating kelp seedlings, characterized in that, Using the kelp endophytic fungus *Priscilla megaterium* as described in any one of claims 1-5 Priestia megaterium H403 is used for kelp seedling cultivation.

10. The kelp seedling cultivation method according to claim 9, characterized in that, The seedling cultivation method specifically includes: [using *Primate* macrocephala...] Priestia megaterium Strain H403 was stored, and the inoculation loop was immersed in the bacterial solution and streaked onto freshly prepared LB agar plates until single colonies grew; *Primate gigantea* was selected using the inoculation loop. Priestia megaterium H403 colonies were obtained and then inoculated into liquid LB medium. The resulting liquid was then centrifuged and washed twice with sterile seawater to obtain the H403 strain. The H403 strain was then added to seawater for kelp seedling cultivation.

Images