Furcellaria sp. and application thereof in preventing and controlling glass shrimp seedlings

Abstract

The present application relates to the field of aquaculture disease prevention and control and microbial technology, and particularly relates to a strain of marine freiland bacteria with quenching function of quorum sensing and application thereof in preventing and controlling glass seedlings of prawn. The strain was preserved in the China General Microbiological Culture Collection Center on July 9, 2026, and the preservation number is CGMCC No.39642. The strain and fermentation supernatant thereof can degrade AHL quorum sensing signal molecules produced by pathogenic bacteria of glass seedlings, weaken the pathogenicity of the pathogenic bacteria without inhibiting the growth of the pathogenic bacteria, and improve the survival rate of the infected prawn seedlings. The strain has good safety, can be applied to Litopenaeus vannamei juvenile culture through water spraying, and can improve the growth performance and disease resistance of prawn juveniles. The present application provides a green biological prevention and control method based on the quorum sensing quenching mechanism for prawn glass seedling diseases.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture disease prevention and control and microbial technology, specifically involving a strain of marine Freelandella with quorum sensing quenching function and its application in the prevention and control of shrimp larvae. Background Technology

[0002] Translucent post-larvae disease (TPD), also known as translucent shrimp larvae disease, is a serious disease that has emerged in shrimp larvae breeding in recent years. It primarily affects larvae and translucent larvae, characterized by rapid onset, rapid spread, and high mortality, causing mass mortality of shrimp larvae in a short period and resulting in severe economic losses to the shrimp larvae breeding industry. Existing research indicates that the occurrence of this disease is closely related to infection by various pathogenic Vibrio bacteria, among which *Vibrio parahaemolyticus*, *V. alginolyticus*, and *V. neocaledonicus* are considered important related pathogens. Therefore, establishing safe, effective, and stable biocontrol technologies for these diseases is of great significance for ensuring the production of *Litopenaeus vannamei* larvae.

[0003] Currently, the control of diseases in shrimp larvae still mainly relies on antibiotics, chemical disinfectants, and environmental control measures. However, long-term or irrational use of antibiotics and chemical agents can easily lead to drug resistance in pathogens and may adversely affect the microecological environment and beneficial microbial communities in aquaculture. Therefore, the use of microorganisms and their metabolic activities for biological control of diseases is gradually gaining attention. Among them, probiotics are considered an important potential technological approach to replace traditional chemical drugs due to their high safety, environmental friendliness, and ability to regulate the aquaculture microecology.

[0004] Existing research has shown that some probiotics can reduce the incidence of aquatic animal diseases by producing antibacterial substances, competing for nutrients and ecological niches, improving the aquaculture environment, or enhancing host immunity. For example, Chinese patent CN114540218B discloses a strain of *Tritonibacter mobilis* R6-3G2, with accession number GDMCC No: 62115. This strain can produce compounds with antibacterial activity, exhibiting antagonistic effects against *Vibrio parahaemolyticus* VP-HL, a pathogen associated with glassy seedlings, and demonstrating effects in reducing pathogen infection and improving shrimp larvae survival rates in shrimp farming experiments. These types of probiotics mainly exert their effects by directly inhibiting or killing pathogens.

[0005] However, obtaining functional probiotics that can be truly applied to the prevention and control of specific aquatic diseases is not easy. On the one hand, the microbial diversity in the aquaculture environment is complex, and different strains exhibit significant differences in physiological characteristics, metabolites, and ecological adaptability. Strains isolated from ordinary environments may not necessarily be able to stably colonize or function in shrimp larvae rearing environments. On the other hand, the in vitro antibacterial activity of antagonistic strains screened against specific pathogens is not directly equivalent to the disease control effect under actual aquaculture conditions. Evaluations at multiple levels, including pathogen antagonism, environmental adaptability, biosafety, and animal infection models, are usually required. Therefore, screening functional probiotics from complex microbial resources that have a clear control effect on diseases affecting shrimp larvae and are suitable for shrimp larvae rearing environments requires establishing a targeted screening system and undergoing multi-stage validation, which presents significant technical challenges.

[0006] Furthermore, current probiotic screening studies largely focus on strains that can directly inhibit pathogen growth, with screening indicators typically based on inhibition zones, minimum inhibitory concentrations (MICs), or pathogen growth inhibition rates. This approach tends to select strains with strong direct inhibitory capabilities as candidates, but it may overlook the distinction between pathogenicity and growth capacity. For diseases affecting glass seedlings, the pathogenic process is not only related to the pathogen's population size but also closely linked to quorum sensing, virulence factor expression, and host-pathogen interactions. Therefore, selectively intervening in the pathogenic process without directly killing or strongly inhibiting pathogen growth holds promise for developing novel biocontrol strategies distinct from traditional antagonistic inhibitory mechanisms.

[0007] Quorum sensing (QS) is a crucial regulatory mechanism by which bacteria sense population density and regulate virulence factors, motility, biofilm formation, and other gregarious behaviors by producing, releasing, and recognizing signaling molecules. Quorum quenching (QQ), on the other hand, blocks or weakens the quorum sensing regulatory process by degrading, modifying, or interfering with pathogenic quorum sensing signaling molecules. Unlike directly killing or inhibiting pathogens, the primary goal of QQ is to reduce the pathogenicity of pathogens rather than directly inhibiting their survival and reproduction, thus providing a new approach for the green control of aquatic diseases.

[0008] However, probiotics with quorum sensing quenching capabilities and effective effects against pathogens causing glassy shrimp infections are not easily screened. Quorum sensing signals are diverse, and the signal generation, recognition, and regulation mechanisms differ among different pathogens. Microorganisms with signal degradation capabilities may only exhibit general signal degradation activity and cannot effectively intervene in the pathogenic process of specific pathogens. Therefore, simply detecting the degradation ability of signal molecules in vitro cannot prove that candidate strains can actually reduce the virulence of glassy shrimp pathogens, let alone prove that they can provide protection in shrimp hosts. To screen QQ functional strains with practical application value from environmental microorganisms, it is usually necessary to sequentially complete signal quenching ability screening, pathogenicity-related phenotypic verification, strain safety and environmental adaptability evaluation, and control effect verification in animal infection models. The screening process is complex, time-consuming, and has a high technical threshold.

[0009] Therefore, current technologies still lack a functional probiotic that can be used for targeted screening of diseases affecting Litopenaeus vannamei larvae, reduce the pathogenicity of related pathogens through indirect antibacterial mechanisms, and demonstrate a clear protective effect in shrimp infection models. In particular, there is a lack of technical solutions for screening functional strains from seawater using quorum sensing quenching mechanisms and further verifying their ability to improve the pathogenicity of pathogens affecting larvae and the survival of shrimp larvae. Therefore, developing a novel probiotic that can be used for targeted screening of diseases affecting larvae, possesses good environmental adaptability and biosafety, and can achieve disease control through indirect antibacterial mechanisms such as quorum sensing quenching is of great significance for expanding green control technologies for diseases affecting shrimp larvae. Summary of the Invention

[0010] The purpose of this invention is to provide a marine *Frederick* strain with quorum sensing quenching function and its application in the control of glassy shrimp larvae. The provided marine *Frederick* strain possesses QQ function, enabling it to efficiently degrade AHL signaling molecules of glassy shrimp pathogens, significantly reducing their virulence without inhibiting pathogen growth. It exhibits good safety for shrimp larvae, and its application via water spraying can effectively promote the growth of Litopenaeus vannamei and improve its disease resistance.

[0011] This invention first provides a strain of *Vreelandella aquamarina* L2M-2, which was deposited on July 9, 2026, at the China General Microbiological Culture Collection Center of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 39642.

[0012] This invention also provides an application of the aforementioned marine Fritillaria L2M-2 strain, which is its use as a quorum sensing quenching strain;

[0013] The present invention also provides the application of the aforementioned marine Fritillaria L2M-2 strain in the preparation of products for the prevention and control of shrimp glassy seedlings.

[0014] The present invention also provides a product for preventing and controlling shrimp larvae from developing glass, which contains live bacteria of the marine strain Frelandia lappa L2M-2.

[0015] Furthermore, the product also contains the fermentation supernatant of the *Frederickia natans* strain L2M-2.

[0016] The *Frederickia fragilis* L2M-2 strain provided by this invention can effectively weaken the virulence of pathogens affecting shrimp larvae through the QQ mechanism without inhibiting pathogen growth, thereby reducing the mortality rate of infected shrimp larvae. This strain has a clear mechanism of action, good safety profile, and is unlikely to induce drug resistance in pathogens, making it a promising candidate for shrimp larvae production. Attached Figure Description

[0017] Figure 1 : Degradation rate of C6-HSL strain tested;

[0018] Figure 2 Phylogenetic tree of target strain L2M-2;

[0019] Figure 3 Results of hemolytic activity of *Frederickia frigidus* strain L2M-2;

[0020] Figure 4 Mortality rate of shrimp larvae infected by immersion in seawater Frylanella L2M-2 strain;

[0021] Figure 5 The degradation effect of seawater Fritillaria cirrhosa strain L2M-2 and its fermentation supernatant on exogenous C6-HSL;

[0022] Figure 6 Effects of L2M-2 strain on QS signaling molecules and growth of 226VH strain in co-culture system;

[0023] Figure 7 Effects of L2M-2 strain on QS signaling molecules and growth of 226VH strain in a mixed system;

[0024] Figure 8 The effect of L2M-2 strain on the pathogenicity of 226VH strain;

[0025] Figure 9 Effects of water spraying with L2M-2 strain on the growth performance of Litopenaeus vannamei larvae;

[0026] Figure 10The effect of water spraying of strain L2M-2 on the disease resistance of Litopenaeus vannamei larvae. Detailed Implementation

[0027] The *Vreelandella aquamarina* strain L2M-2 screened in this invention was obtained from the water of a healthy Litopenaeus vannamei pond.

[0028] The *Frederickia natans* L2M-2 strain provided by this invention possesses QQ (qq) function. Its bacterial culture and fermentation supernatant can efficiently degrade exogenously added AHL signaling molecule C6-HSL and naturally occurring AHL signaling molecules in the culture system of *Vibrio 226VH* (New Caledonian Vibrio), without inhibiting the growth of *Vibrio 226VH*. Furthermore, this *Frederickia natans* L2M-2 strain exhibits good safety.

[0029] The present invention will now be described in detail with reference to the embodiments and accompanying drawings. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] The formulation of the minimal salt medium (MSM) used in the examples is as follows:

[0031] 100 mL of pure water, 3 g of sea salt, 0.05 g of NaHCO3, 0.1 g of (NH4)2SO4, pH 7.0~7.5, sterilized, then add 0.1 mL of sterile trace element stock solution.

[0032] The formula for trace element mother liquor is as follows:

[0033] 1000 mL pure water, 1 g FeSO4·7H2O, 0.45 g MnSO4·H2O, 0.44 g ZnSO4·7H2O.

[0034] The LB medium used has the following components:

[0035] 1000 mL pure water, 10 g tryptone, 5 g yeast powder, 30 g NaCl, pH 7.2-7.4.

[0036] Example 1: Optimization of quorum induction quenching sterilization screening method and strain identification

[0037] 1) Quorum induction quenching sterilization screening method

[0038] Water samples were collected from healthy shrimp ponds in Qingdao, Weifang, and Yancheng shrimp farms from June to July 2025. To avoid the impact of eutrophic media on the aquatic microecology, an oligotrophic medium was optimized for enriching and isolating the target bacterial strains in the water. 1 mL of sample was centrifuged at 6000 × g for 10 min, the precipitate was collected, resuspended in 1 mL of MSM-AHL (C6-HSL was selected as the exogenous AHL additive at a concentration of 20 μg / mL), and 10% 1,4-piperazinediethanesulfonic acid (PIPEs) buffer was added. The mixture was then incubated in a constant temperature shaker (30 ℃, 180 r / min) for 24 h. The culture was then subcultured into fresh MSM-AHL liquid at a 1% inoculum rate, for a total of four subcultures. After the final subculture, the bacterial culture was plated onto LB solid medium to isolate and purify single colonies.

[0039] After culturing purified single colonies in LB medium for 24 h, 178 μL of the bacterial culture was mixed with 2 μL of C6-HSL (10 μg / mL) and 20 μL of PIPEs buffer to prepare an incubation system. The culture was incubated for 24 h (30 ℃, 180 r / min), then centrifuged (10280×g, 10 min) and 10 μL of the supernatant was collected. Agrobacterium tumefaciens strain A136 was used as an indicator strain for AHL signaling molecules. 5-bromo-4-chloro-3-indole-β-D-galactoside (X-gal) was added to the A136 culture system to a final concentration of 250 μg / mL and incubated for 24 h (30 ℃, 180 r / min). Then, 190 μL of the mixture was mixed with the 10 μL of supernatant and reacted for 12 h (30 ℃). Finally, the OD of the mixture was measured using a microplate reader. 630 and OD 490 And through the formula (0.653×OD) 490 -OD 630 ) / (0.267×OD 630 -OD 490 The β-gal activity in the system was calculated, and higher activity indicated a higher concentration of AHL signaling molecules in the mixed system.

[0040] Results: By resuspending and continuously subculturing the bacterial sediment in oligotrophic medium, bacteria with AHL degradation function in the environment could be accurately screened. A total of 40 pure colonies were isolated from three batches of water samples, and all of them were found to have a certain degree of AHL degradation function (see Table 1).

[0041] Table 1: Screening Results of Quenching Bacteria by Quenching

[0042] NC 0.10 ± 0.01* L3-3 0.23 ± 0.04* S2-6 0.26 ± 0.02* C2-2 0.19 ± 0.03* PC 0.40 ± 0.02 L3M-2 0.23 ± 0.02* C2-1 0.18 ± 0.02* C2-3 0.11 ± 0.01* L1-1 0.23 ± 0.02* S1-2 0.32 ± 0.03* C8-2 0.24 ± 0.04* C2-4 0.25 ± 0.03* L1M-1 0.23 ± 0.02* S1-3 0.32 ± 0.07* C8-3 0.31 ± 0.02* C2-5 0.15 ± 0.02* L1M-2 0.27 ± 0.04* S1-4 0.17 ± 0.01* C8-4 0.25 ± 0.01* C5-1 0.22 ± 0.01* L1M-3 0.32 ± 0.02* S1-5 0.14 ± 0.01* C8-5 0.21 ± 0.01* C5-2 0.14 ± 0.01* L2M-1 0.24 ± 0.02* S2-1 0.31 ± 0.08* C12-1 0.22 ± 0.02* C5-3 0.17 ± 0.01* L2M-2 0.19 ± 0.02* S2-2 0.23 ± 0.04* C12-2 0.27 ± 0.01* C5-4 0.22 ± 0.01* L2M-3 0.23 ± 0.01* S2-3 0.14 ± 0.01* C12-3 0.26 ± 0.01* C8-1 0.14 ± 0.02* L3-1 0.26 ± 0.02* S2-4 0.16 ± 0.01* C12-4 0.24 ± 0.00* L3-2 0.25 ± 0.02* S2-5 0.17 ± 0.07* C12-5 0.30 ± 0.03*

[0043] Note: NC is the negative control group, with no added bacterial culture or signaling molecules; PC is the positive control group, with only added signaling molecules. * indicates a significant difference compared to the PC group (P ≤ 0.05).

[0044] 2) Detection of C6-HSL signal molecule degradation rate of the test strain

[0045] Strains exhibiting good AHL signal molecule degradation were selected as test strains, and their degradation rate was measured. The AHL signal molecule concentration detection method was as described above, with the addition of several groups of sterile systems with different concentrations of C6-HSL to create a standard curve. By constructing the standard curve, the remaining C6-HSL signal molecule concentration in the system after treatment with each test strain was calculated, and the degradation rate was also calculated.

[0046] Further analysis of the 13 most effective strains (β-gal activity ≤ 0.20) revealed that the strains with the highest C6-HSL signaling molecule degradation rates were L2M-2, S1-5, S2-3, C2-2, C2-3, and C5-2 (see [link to relevant documentation]). Figure 1 ).

[0047] 3) Strain identification

[0048] Identification Method: The above six bacterial strains were selected for 16S rDNA sequencing identification. The six test strains were inoculated onto the surface of LB medium using a sterile inoculation loop and cultured at 30 °C for 24 h. Genomic DNA was then extracted from the six test strains using the Ezup column-based bacterial genomic DNA extraction kit (Sangon Biotech, Shanghai, China), and the 16S rDNA gene was amplified. The primer nucleotide sequences used were 27F (5'-AGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTT ACGACTT-3'). The 16S rDNA sequences of the six test strains were compared with those in NCBI (https: / / blast.ncbi.nlm.nih.gov) and the Ribosome Database (http: / / rdp.cme.msu.edu / index.jsp), and a phylogenetic tree of the target strains was constructed using MEGA 11 software.

[0049] Results: Comparison with the NCBI database revealed that, among the six tested strains, L2M-2 showed the highest similarity to *Frederickia natans*. Of the remaining five strains, two were Vibrio, one was *Alteromonas simiduii*, and two were *Pseudomonas abyssi*, all of which are pathogenic or opportunistic pathogens in aquaculture (see Table 2). Therefore, L2M-2 was selected as the target strain.

[0050] Table 2: Results of 16S rDNA Identification of Test Strains

[0051] L2M-2 Vreelandella aquamarina S1-5 Vibrio sp. S2-3 Vibrio sp. C2-2 Alteromonas simiduii C2-3 Pseudomonas abyssi C5-2 Pseudomonas abyssi

[0052]

[0053] Based on pattern strain comparison and phylogenetic tree construction using ribosomal databases, strain L2M-2 (GenBank accession number: PZ610728) was identified as belonging to the genus *Frenchella*, exhibiting the highest similarity to *Frenchella marinei* (see [link to relevant documentation]). Figure 2 ).

[0054] Example 2: Safety assessment of the strain

[0055] The in vivo and in vitro safety assessments of the marine Fritillaria cirrhosa strain L2M-2 were conducted, specifically involving hemolytic assays, antibiotic susceptibility assays, whole genome sequencing, and immersion infection experiments.

[0056] Hemolytic activity test: When the L2M-2 bacterial suspension cultured in LB medium is adjusted to the desired concentration, 10 μL of the bacterial suspension is dropped onto a sterile filter paper on the surface of blood agar medium. After incubation at 30 ℃ for 24 h, observe whether a hemolytic zone is formed.

[0057] Antibiotic susceptibility testing: Antibiotic susceptibility testing strips from Hangzhou Microbial Reagent Co., Ltd. were used. These strips were then affixed to surfaces uniformly coated with L2M-2 bacterial suspension (OD200). 600 On LB solid medium (= 1.0), after incubation at 30 ℃ for 24 h, the diameter of the inhibition zone was measured and drug sensitivity was determined according to the CLSI 2011 standard.

[0058] Whole-genome sequencing: Genomic DNA was extracted from the logarithmically growing strain L2M-2 using the STE method. Integrity was checked and quantified by agarose gel electrophoresis and a Qubit fluorometer. After passing quality control, whole-genome sequencing was performed by Beijing Novogene Bioinformatics Technology Co., Ltd. using single-molecule real-time (SMRT) technology on the PacBio Sequel platform, constructing a 10kb insert library. After the raw sequencing data was processed, low-quality reads were filtered using SMRT Link software (v8.0). Subsequently, the filtered high-quality data was assembled de novo using Canu software (v2.0, https: / / github.com / marbl / canu) to generate a complete genome sequence without gaps. After assembly, basic characteristics such as genome size, GC content, chromosome and plasmid composition, and circularity were analyzed. GeneMarkS software (http: / / exon.gatech.edu / GeneMark / ) was used to predict coding genes and count the total number of genes. The EffectiveT3 software (https: / / effectors.csb.univie.ac.at) was used to predict proteins in secretory systems of types I-VII, and the number of genes in each type of secretory system was counted. The predicted coding gene sequences were then BLAST-aligned with the virulence factor database VFDB (http: / / www.mgc.ac.cn / VFs) (E-value < 1e-5). Based on the annotation results, the number of high-risk pathogenic genes (toxins, hemolysins, invasive agents, etc.) and medium-risk helper virulence factors (membrane-associated factors, motion / adhesion factors, metabolic-associated factors, etc.) was counted.

[0059] Immersion infection experiment: Healthy Litopenaeus vannamei larvae at stage P12 were selected as experimental materials. Three groups were set up: the NC group received an equal volume of sterile saline, and the L2M-2 group received a final concentration of 1×10⁻⁶. 5 CFU / mL of L2M-2 strain bacterial suspension was added, with strain 226VH of glassy shrimp pathogens as the PC group, at the same concentration as L2M-2 strain. Each group had three replicates, with 50 shrimp larvae stocked in 20 L of water (salinity 20) per replicate. No feeding was performed during the experiment; dissolved oxygen was ≥ 5 mg / L; the average water temperature was 27.8℃; and observation continued for 96 h, recording the mortality rate of the tested shrimp larvae.

[0060] Results: The L2M-2 strain exhibited γ-hemolytic activity on blood agar plates, i.e., it was non-hemolytic (see...). Figure 3 The L2M-2 strain showed resistance to four antibiotics, including penicillin, moderate sensitivity to erythromycin, and sensitivity to 15 antibiotics, including carbenicillin (see Table 3).

[0061] Table 3: Antibiotic susceptibility results of Marine Frylanella L2M-2 strain

[0062] Penicillin (P) 10 U 11.41 ± 1.49 R Oxacillin (OX) 1 μg 10.39 ± 0.95 R Ampicillin (AM) 10 μg 11.30 ± 2.80 R Carbenicillin (CB) 100 μg 20.51 ± 3.16 S Piperacillin (PIP) 100 μg 13.95 ± 1.75 R Cephalexin (CA) 30 μg 20.95 ± 0.45 S Cefamezin (CZ) 30 μg 36.43 ± 2.25 S Cefradine (RAD) 30 μg 23.30 ± 0.52 S Cefuroxim (CXM) 30 μg 27.49 ± 1.28 S Ceftazidime (CAZ) 30 μg 27.89 ± 1.67 S Ceftriaxone (CTR) 30 μg 29.47 ± 0.36 S Cefoperazone (CFP) 75 μg 22.58 ± 0.68 S Amikacin (AK) 30 μg 18.13 ± 0.54 S Gentamicin (GM) 10 μg 19.14 ± 1.57 S Kanamycin (K) 30 μg 18.56 ± 1.62 S Neomycin (N) 30 μg 17.77 ± 0.49 S Tetracycline (TE) 30 μg 18.50 ± 1.10 S Doxycycline (DX) 30 μg 19.23 ± 1.52 S Minocycline (MI) 30 μg 19.69 ± 0.58 S Erythromycin (E) 15 μg 18.24 ± 2.09 I

[0063] Note: R indicates drug resistance, I indicates moderate sensitivity, and S indicates sensitivity.

[0064] The L2M-2 strain contains one circular chromosome and has a total genome length of 3,877,170 bp (see Table 4).

[0065] Table 4: Basic Information on Chromosomes and Plasmids of Marine Frylanella L2M-2 Strain

[0066] L2M-2 Chromosome Chr1 3877170 58.16 circular

[0067] The statistical results of secretion system-related genes are shown in Table 5. Strain L2M-2 contains only one T6SS secretion system; the key virulence factor secretion system T3SS was not detected. Virulence factor database annotation results showed that no known high-risk pathogenic genes (such as toxins, hemolysins, invasive agents, etc.) were annotated. The 83 detected intermediate-risk factors were all accessory functions related to membranes, motility / adhesion, and metabolism. These functions are crucial for the intestinal colonization and survival of probiotics and are not virulence markers (Table 6).

[0068] Table 5: Gene distribution of the secretory system of *Frenchia frenulum* strain L2M-2

[0069] L2M-2 3611 0 0 0 0 0 1 0

[0070] Table 6: Annotation results of the Virulence Factor Database (VFDB) for Marine Fritillaria L2M-2 strain

[0071] Total number of effectively annotated genes 261 High-risk pathogenic genes (toxins / hemolysins / invasive substances, etc.) 0 Total number of medium-risk auxiliary virulence factors 83 Types and number of medium-risk factors Membrane-related factors: 46; T6SS: 2; Motility / adhesion factors: 31; Metabolic factors: 4 Genes unrelated to pathogenicity 178

[0072] In the immersion infection test, no shrimp larvae in the L2M-2 group showed mortality, and the survival rate was significantly higher than that in the PC group (see...). Figure 4 Therefore, strain L2M-2 is safe for Litopenaeus vannamei larvae and can be used in shrimp larvae breeding and production.

[0073] Example 3: Verification of Quorum Sensing Quenching Activity of Strains

[0074] Assay for AHL degradation function of supernatant: *Frederickia natans* strain L2M-2 was cultured in LB medium for 24 h, centrifuged at 8000 r / min for 10 min, and the supernatant was filtered through a 0.22 μm sterile membrane to prepare sterile fermentation broth supernatant L2M-2S. 178 μL of bacterial culture and sterile supernatant were respectively mixed with 2 μL C6-HSL (5 mmol / L) and 20 μL PIPEs buffer to prepare incubation systems. *Agrobacterium tumefaciens* strain A136 was used as an indicator bacterium to detect the concentration of signal molecules in each system. The NC group did not add any bacterial culture, supernatant, or signal molecules, while the PC group used 178 μL of sterile medium instead of bacterial culture and supernatant.

[0075] Co-culture experiment: The concentrations of the cultured glass seedling pathogen strain 226VH and marine Fritillaria cirrhosa strain L2M-2 were adjusted to OD. 600 =1, and then both strains were co-inoculated into the same new LB medium at an inoculum volume of 1%. The control group was inoculated with only strain 226VH, and a co-culture group of strain 226VH and C6-HSL at a final concentration of 2.5 μg / mL was set up. After culturing for 24 h (30 ℃, 180 r / min), the concentration of AHL signal molecules in each co-culture system was detected using strain A136, and the density of strain 226VH in each co-culture system was detected by TCBS agar plate spreading method.

[0076] Mixed experiment: The concentrations of the cultured 226VH strain and L2M-2 strain were adjusted to OD. 600 =1, and prepare sterile fermentation supernatant L2M-2S for strain L2M-2. A 1:1 mixing ratio was set, i.e., 90 μL of L2M-2 bacterial culture and sterile supernatant were each mixed with 90 μL of 226VH bacterial culture in 2 mL sterile centrifuge tubes, and then 20 μL of PIPEs buffer was added to prepare a 200 μL mixture. The control group consisted of 226VH strain mixed with an equal volume of sterile culture medium, and a separate 200 μL mixture of 226VH strain and C6-HSL (final concentration 2.5 μg / mL) was prepared. The concentration of AHL-type signal molecules in each co-culture system was detected using strain A136, and the density of 226VH strain in each co-culture system was detected using the TCBS agar plate method.

[0077] Results: Both the bacterial culture and sterile fermentation supernatant of strain L2M-2 effectively degraded exogenously added C6-HSL signaling molecules (see [link to results]). Figure 5 In co-culture experiments, exogenous addition of C6-HSL signaling molecules led to an increase in the concentration of AHL signaling molecules in the system but did not affect the growth of strain 226VH. However, when strains L2M-2 and 226VH were present in the environment, the growth of strain 226VH was not affected, but the concentration of AHL signaling molecules in the system decreased significantly (see...). Figure 6Similar phenomena were observed in the mixed experiments: contact between L2M-2 bacterial culture and sterile fermentation supernatant and 226VH strain significantly reduced the concentration of AHL signaling molecules in the environment, but the density of 226VH strain was not affected by the concentration of AHL signaling molecules in the environment (see...). Figure 7 ).

[0078] The above results indicate that the presence of L2M-2 strain or its fermentation supernatant in the environment will inhibit the QS system of the glass seedling pathogen, but will not affect its growth.

[0079] Example 4: The weakening effect of the strain on the virulence of the pathogen of glass seedlings

[0080] The effect of L2M-2 strain on the pathogenicity of 226VH strain: The experiment was conducted using the immersion infection method. Healthy Litopenaeus vannamei larvae were selected, with a body length of (1.477±0.402) cm and a weight of (0.029±0.019) g. Three groups were set up: the NC group (with an equal volume of sterile saline), the 226VH group, and the L2M-2+226VH group. Each group had three replicates, with 15 larvae placed in 1 L of water in each replicate. The cultured L2M-2 and 226VH strains were adjusted to OD. 600 After the values ​​were consistent, the mixture was prepared at a 1:1 volume ratio and allowed to react for 24 h. The 226VH group served as a control, with an equal volume of 226VH bacterial suspension mixed with sterile LB medium. Subsequently, shrimp larvae were infected with the same 226VH concentration to achieve a final concentration of 1×10⁻⁶ in the water. 6 CFU / mL. During the infection period, no feeding was provided, and continuous oxygenation was maintained at dissolved oxygen ≥5 mg / L, water temperature (28.70±0.26)℃, salinity (19.00±1.73), and pH (8.49±0.07). The disease incidence and mortality of shrimp larvae in each group were recorded continuously for 96 h.

[0081] The effect of L2M-2 strain supernatant on the pathogenicity of 226VH strain: Using the same method as above, another batch of Litopenaeus vannamei larvae was used for a repeat verification experiment. The larvae had a body length of (2.220±0.437) cm and a weight of (0.087±0.050) g. In this batch of experiments, the aseptic fermentation supernatant of L2M-2 strain (L2M-2S) was used to replace the L2M-2 strain bacterial solution and was mixed with the 226VH strain bacterial solution at a 1:1 volume ratio for 24 h. The experiment included an NC group, a 226VH group, and an L2M-2S+226VH group, with 3 replicates in each group. In each replicate, 15 larvae were placed in 1 L of water. The infection method and the final concentration of 226VH were the same as above. During the infection period, no feeding was provided, and continuous oxygenation was maintained at dissolved oxygen ≥5 mg / L, water temperature (28.40±0.10)℃, salinity (20.67±1.15), and pH (8.46±0.02). Observations were conducted continuously for 96 hours, and the morbidity and mortality of shrimp larvae in each group were recorded.

[0082] Results: Both the bacterial culture of strain L2M-2 and its fermentation supernatant significantly reduced the pathogenicity of strain 226VH and significantly improved the survival rate of infected shrimp larvae after contact with strain 226VH (see [link to results]). Figure 8 ).

[0083] Example 5: Effects of water spraying with L2M-2 strain on the growth and disease resistance of Litopenaeus vannamei larvae

[0084] Culture Experiment: Healthy Litopenaeus vannamei larvae were selected, with a body length of (1.087±0.178) cm and a weight of (0.006±0.001) g. Two groups were set up: the NC group and the L2M-2 group, with three replicates in each group. Each replicate stocked 250 Litopenaeus vannamei larvae in 100 L of water. L2M-2 bacterial solution was sprayed into the culture water of the L2M-2 group daily until the final concentration reached 1×10⁻⁶. 5 CFU / mL, the NC group was treated with an equal volume of sterile saline solution daily. The culture experiment lasted for 21 days, with feed administered twice daily (9:00 and 17:00) at a basal daily intake of 15%, adjusted according to feeding behavior. Both groups were fed the same amount. During the culture period, the water temperature was (28.60±0.48)℃, salinity was (18.50±0.76), pH was (8.47±0.05), and dissolved oxygen was ≥5mg / L. After the culture ended and feeding was stopped for one day, the body length and weight of shrimp larvae in each group were measured, and the relative weight gain rate and specific growth rate were calculated to evaluate the effect of strain L2M-2 on the growth performance of shrimp larvae.

[0085] Challenge Experiment: After the culture period, uniformly sized shrimp larvae from each group were selected for a bath challenge experiment. The experiment consisted of three groups: the NC group, the 226VH group, and the L2M-2 group, with three replicates per group. Each replicate consisted of 15 shrimp larvae placed in 1 L of water. The larvae used in the NC and 226VH groups were derived from the NC group in the culture experiment. An equal volume of sterile saline was added to the NC group, while 226VH strain bacterial solution was added to the water in the PC and L2M-2 groups, with a final concentration of 1×10⁻⁶. 6 CFU / mL. During the infection period, no feeding was provided, and continuous oxygenation was maintained at dissolved oxygen ≥5 mg / L, water temperature (29.30±0.20)℃, salinity (21.00±0.07), and pH (8.36±0.04). Observations were conducted every 12 hours, and the morbidity and mortality of shrimp larvae in each group were recorded.

[0086] The results are as follows Figure 9 As shown, strain L2M-2 effectively improved the growth performance of Litopenaeus vannamei larvae, with significantly higher terminal body length, body weight, relative weight gain, and specific growth rate compared to the NC group. Furthermore, after infection with pathogen strain 226VH, strain L2M-2 significantly improved the 48-hour survival rate of shrimp larvae (see...). Figure 10 The results are consistent with those in Example 4.

Claims

1. A marine Fritillaria cirrhosa bacterium, characterized in that, The preservation number of the *Frederickia natans* is CGMCC No. 39642.

2. The application of the *Frederickia natans* strain described in claim 1 as a quorum sensing quenching functional strain.

3. The use of the marine Freeland bacteria as described in claim 1 in the preparation of products for the prevention and control of shrimp larvae.

4. A product with the effect of preventing and controlling glassy larvae in shrimp, characterized in that, The product contains live bacteria of the marine strain Frelandia lappa L2M-2.

5. The article of claim 4, characterized in that, The product further comprises the fermentation supernatant of *Frederickia natans* as described in claim 1.

6. The application of the marine Frevanobacterium as described in claim 1 in promoting the growth and development of shrimp.

7. A method for promoting shrimp growth, characterized in that, The method involves adding the product described in claim 4 to the shrimp farming water.

Images