Microbial agent for preventing and treating porphyra red rot and preparation method thereof

Abstract

The invention discloses a microbial agent for preventing and treating porphyra red rot and a preparation method thereof, and belongs to the technical field of seaweed disease prevention and treatment. The microbial agent comprises a Pseudoalteromonas piscicida strain P3 (the preservation number is CCTCC (China Center for Type Culture Collection) No. M20221357) or a P6 (the preservation number is CCTCC No. M20221356), and a fermentation liquid of the Pseudoalteromonas piscicida strain P3 or the P6 and the fermentation liquid of the Pseudoalteromonas piscicida strain P6 and the fermentation liquid of the Pseudoalteromonas piscicida strain P6. The invention also discloses an optimized fermentation process of the microbial agent and a synergistic prevention and control method of biological soaking and physical drying. During application, the laver is soaked in 106-108 CFU / mL of the bacterial liquid for 3 seconds to 3 minutes, and then is subjected to drying treatment for 0.5-1.5 hours. The strain screened by the invention has broad-spectrum and strong antagonistic activity on a plurality of porphyra pythium, and is safe and harmless to the growth of porphyra. Experiments show that the disease spot inhibition rate can reach 77.23%-86.51% by adopting the P6 strain in cooperation with dry-out treatment, complementary advantages of biological control and physical dry-out are achieved, and the P6 strain has the advantages of being efficient, safe, environmentally friendly and the like and has wide industrial application prospects.

Description

Technical Field

[0001] This invention relates to the field of laver disease control, specifically to a microbial agent for controlling laver red rot and its preparation method. Background Technology

[0002] Red rot is one of the major diseases during the cultivation of laver. It was first reported by Japanese scholar ARASAKI in 1947. When laver is infected with Pythium sibiricum, the thallus activates the algal defense system by accumulating reactive oxygen species (ROS), increases the activity of defensive enzymes such as polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL) in laver cells, and synthesizes a large amount of proline (Pro) to maintain osmotic pressure balance in cells (Zhang Xiaonan, 2015).

[0003] Currently, the main methods for controlling red rot disease in laver include physical control, chemical control, and biological control.

[0004] The physical control method for red rot disease in laver mainly combines drying the laver using netting with cold storage. Laver thallus possesses biological characteristics such as drought and low-temperature tolerance. The cold storage netting technology for *Porphyra yezoensis* was widely used in Japan as early as the 1960s and is an effective means of controlling laver diseases and improving laver yield and quality (Wang Hanqing et al., 1997; Ma Jiahai et al., 1998; Weng Lin, 2007). Cold storage can prevent cultivated laver from experiencing disease outbreaks and has a certain inhibitory effect on the spread of red rot, but it cannot completely kill the pathogen.

[0005] The chemical control method for red rot disease in laver is acid washing. Acid washing utilizes the biological characteristics of laver's tolerance to low acidity and the fact that Pythium mold is not, thus killing Pythium mold and achieving the purpose of preventing red rot disease. However, long-term use of acidic fungicides will increase the acid tolerance of Pythium mold, leading to a decrease in the control effect.

[0006] Biological control techniques for red rot disease in laver are still in the preliminary laboratory research stage. KITAMURA et al. isolated two strains each of Bacillus and Pseudomonas that can degrade the cell walls of Pythium spp. in laver (KITAMURA et al., 2002). WOO et al. discovered an antimicrobial protein, SAP, in the culture supernatant of marine Streptomyces that can inhibit the growth of Pythium spp. This protein can cause swelling and disintegration of Pythium hyphae without affecting the survival rate of laver cells. WOO et al. speculated that its anti-Pythium mechanism is related to altering the membrane permeability of Pythium spp. (WOO et al., 2002; WOO et al., 2003). DING et al. isolated a strain of Cladosporium N5 that can produce the growth regulators phenylacetic acid and p-hydroxyphenylacetic acid for Laveria bassiana, and no cytotoxicity was found in lethality tests on marine shrimp. It can be used as a potential biocontrol bacterium to protect algae from pathogens (DING et al., 2008). VALLET et al. isolated several pyranone compounds from *Pythium spp.* strain AN596H, which can inhibit infection of *Pythium spp.* and *Pythium spp.* on *Porphyra* (VALLET et al., 2018). Zou Dandan screened 40 bacteria with antagonistic ability against *Pythium* mycelial growth, among which the fermentation broths of strains 188, A6, and O also inhibited *Pythium spp.* mycelial growth (Zou Dandan, 2016).

[0007] Although there are currently various methods for preventing and controlling red rot disease in seaweed, none of them are truly efficient, safe, healthy, or economical. Summary of the Invention

[0008] The purpose of this invention is to provide a microbial agent for preventing and controlling red rot disease in laver and its preparation method, and to establish a highly efficient prevention and control process that combines biological soaking and physical drying.

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] This invention discloses a microbial agent for preventing and controlling red rot disease in laver, comprising Pseudoalteromonas piscicida strain P3, strain P6 or their fermentation broth.

[0011] Preferably, strain P3 is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with accession number CCTCC No. M20221357 and accession date of August 31, 2022; strain P6 is deposited at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC No. M20221356 and accession date of August 31, 2022.

[0012] Preferably, the above-mentioned fermentation broth preparation method is as follows:

[0013] a. Preparation of fermentation culture medium

[0014] The optimized 2216E modified culture medium was used, and its components are as follows:

[0015] The following ingredients were prepared: 5.0 g / L peptone, 1.0 g / L yeast extract, 0.1 g / L ferric citrate, 19.45 g / L sodium chloride, 5.9 g / L magnesium chloride, 3.24 g / L magnesium sulfate, 1.8 g / L calcium chloride, 0.55 g / L potassium chloride, 0.16 g / L sodium bicarbonate, 0.08 g / L potassium bromide, and trace amounts of strontium salts and borates. The solution was prepared with aged seawater or artificial seawater, and the pH was adjusted to 7.6-7.8. The solution was then autoclaved at 121°C for 20 min.

[0016] b. Strain activation and seed culture preparation

[0017] (1) Slant activation: Inoculate the antagonistic bacteria P3 or P6 stored in glycerol tubes at -80℃ onto 2216E agar plates and incubate at 28℃ for 24-48 h. Select single colonies with neat edges and full shape.

[0018] (2) Primary seed culture: The activated single colony was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of 2216E liquid medium and cultured with shaking at 28℃ and 150 r / min for 12-18 h until the bacterial concentration reached OD600 of 1.0-1.2 to obtain the primary seed culture.

[0019] (3) Secondary seed culture (optional): Transfer the primary seed culture to a larger volume of the same culture medium at an inoculation rate of 5%-10%, and culture under the same conditions for 10 h to obtain a highly viable seed culture.

[0020] c. Fermentation process in fermentation tanks

[0021] Inoculate the above seed culture into the fermenter at an inoculum volume of 5%-8% (filling the fermenter to 60%-70%). Control the fermentation parameters as follows:

[0022] Fermentation temperature: 26-28℃;

[0023] Stirring speed: Initial speed 150 r / min, adjusted according to dissolved oxygen level to maintain dissolved oxygen (DO) at 30%-50%;

[0024] Ventilation rate: 1.0-1.5 vvm;

[0025] Defoaming control: Automatic addition of silicone defoamer;

[0026] Fermentation time: Continue fermentation for 48-72 hours;

[0027] During fermentation, samples were taken every 6 hours to test cell concentration and pH value. Fermentation was stopped when cell growth reached a plateau and metabolite concentrations reached their peak.

[0028] d. Post-processing of fermentation products

[0029] Whole-culture fermentation broth: The mixture after fermentation is complete is the whole-culture fermentation broth, at which point the bacterial concentration should reach 10. 9 -10¹ 0 CFU / mL.

[0030] Sterile filtrate (metabolites): To prepare a sterile filtrate, centrifuge the fermentation broth at 4°C and 8000 r / min for 15 min, discard the cell precipitate, and collect the supernatant. Filter the supernatant through a 0.22 μm microporous membrane for sterilization; the resulting liquid is the sterile filtrate containing antagonistic active substances (such as antimicrobial proteins or secondary metabolites).

[0031] e. Dilution before application

[0032] In the actual control of red rot disease in laver, the above fermentation broth or filtrate is diluted with sterilized seawater to an effective concentration (e.g., 10) according to experimental requirements. 7 (CFU / mL) can be used for soaking or spraying seaweed.

[0033] This invention also provides a method for preventing and controlling red rot disease in laver, which involves soaking laver in the aforementioned microbial preparation.

[0034] Preferably, the concentration of *Pseudomonas pseudoalteromonas* in the above-mentioned microbial preparation is 10. 6 -10 8 CFU / mL.

[0035] Preferably, the above method also includes drying the laver after soaking.

[0036] Preferably, the soaking time is 3 seconds to 3 minutes; the drying time is 0.5 hours to 1.5 hours.

[0037] Preferably, the above method uses a concentration of 10 7 The seaweed was soaked in a solution of strain P6 with CFU / mL for 3 minutes, and then dried for 1 hour.

[0038] Compared with the prior art, the present invention has the following significant advantages:

[0039] 1. Highly effective antagonistic: The strains P3 and P6 screened in this invention exhibit broad-spectrum and strong anti-Pythium activity at 15°C (the temperature at which laver is susceptible to disease) and 24°C (the optimal growth temperature of Pythium), and can effectively inhibit a variety of Pythium strains, including Pythium porphyrae and Pythium chondricola.

[0040] 2. High safety: Safety evaluations show that high concentrations (10) are safe. 7 Treatment with strains P3 and P6 (CFU / mL) did not cause oxidative damage to seaweed cells (ROS and MDA levels returned to normal), did not affect the photosynthetic efficiency (Fv / Fm) of seaweed, and did not cause seaweed to rot or inhibit growth.

[0041] 3. Technological Innovation: This invention is the first to propose a synergistic control strategy of "biological control + physical drying". Experimental data show that short-term drying alone (1 hour) has no significant inhibitory effect on red rot, but when combined with soaking in P6 bacterial solution, it can significantly improve the lesion inhibition rate, realizing the complementary advantages of physical and biological methods. Attached Figure Description

[0042] Figure 1 The growth of the antagonistic bacteria described in this invention is shown in the initial screening results; wherein, A. blank control; B. negative control group; C. Pythium spp. grows on a culture medium containing sterile filtrate of P3(C), P6(D), and P19(E).

[0043] Figure 2 The inhibition rate of the antagonistic bacteria against Pythium growth described in this invention is: AC represents the inhibition rate of P3(A), P6(B), and P19(C) against Pythium at 15℃ and 24℃, and ac represents repeated experiments; DE represents the inhibition rate of the antagonistic bacteria against Pythium growth at 24℃(D) and 15℃(E), and de represents repeated experiments.

[0044] Figure 3 This is the Neighbor-joining phylogenetic tree constructed based on 16S rRNA-dnaA-dnaN-recA of antagonistic bacteria as described in this invention;

[0045] Figure 4 This is a photograph showing the confrontation between 8 strains of Pythium and 3 antagonistic bacteria under 24°C conditions according to the present invention.

[0046] Figure 5 This is a photograph showing the confrontation between 8 strains of Pythium and 3 antagonistic bacteria under 15°C conditions according to the present invention.

[0047] Figure 6 The results of ROS and MDA enzyme activity assays in this invention are shown, wherein: A represents the ROS assay result; B represents the MDA assay result;

[0048] Figure 7 The changes in chlorophyll fluorescence parameters of laver after soaking in bacterial solution are shown in the figure. In this figure: A is the result of Fv / Fm (maximum photochemical quantum yield); B is φPSII (photochemical quantum yield).

[0049] Figure 8 This is a statistical chart showing the growth rate of *Porphyra tenuifolia* according to the present invention;

[0050] Figure 9 These are macroscopic photographs of laver cells and laver after 12 days of soaking with the antagonistic bacteria of the present invention; where a~c. are laver cells of the control group (a), the antagonistic bacteria P3 soaking group (b), and the antagonistic bacteria P6 soaking group (c); A~C are laver cells of the control group (A), the antagonistic bacteria P3 soaking group (B), and the antagonistic bacteria P6 soaking group (C); the scale bar is 1 cm;

[0051] Figure 10 To demonstrate the antagonistic effect of the antagonistic bacteria against Pythium infection in this invention, where: N is the blank control group; P is the positive control group; A and B are the groups of laver treated with P3(A) and P6(B) in advance; CF are the groups of laver treated with P3(C) and P6(D) after 24 h of Pythium in advance and with P3(E) and P6(F) after 72 h; the scale bar is 1 cm.

[0052] Figure 11 To illustrate the efficacy of the antagonistic bacterium P6 in treating red rot disease of laver, the following data are presented: N represents the blank control; P represents the positive control group of laver; d represents the uninfected laver group soaked in P6 bacterial solution for 3 min and then dried for 1 h; O represents the laver group dried for 1 h; AD represents the laver groups soaked in P6 bacterial solution for 3 s (A), 30 s (B), 1 min (C), and 3 min (D) and then dried for 1 h; the scale bar is 1 cm. Detailed Implementation

[0053] Example 1

[0054] Screening of antagonistic bacteria

[0055] The collected samples were placed in sterile sampling bags (Haibo Biotechnology) and water sample collection bags (Haibo Biotechnology) and brought back to the laboratory for processing. The filamentous shells of *Porphyra yezoensis* were rinsed three times with sterile seawater. After flaming and cooling a scraper with an alcohol lamp, approximately 1 cm² of the shell filaments were scraped off. The leaf-like parts of *Porphyra yezoensis* and kelp seedlings were rinsed three times with sterile seawater to remove surface impurities. Approximately 0.2 g of each processed sample was placed in a 1.5 mL sterile EP tube and homogenized using a sterile grinding rod. The resulting homogenates were serially diluted with sterile seawater, and 100 μL of each dilution (10⁻², 10⁻³, 10⁻⁴) was spread onto Zobell 2216E agar. The collected water samples were diluted to 10⁻¹, 10⁻², and 10⁻³, and 100 μL was spread onto Zobell 2216E agar. All plates were inverted and incubated at 28°C for 2 days. Two days later, single colonies with different morphologies were picked from each plate and purified on Zobell 2216E agar medium for later use.

[0056] Take a Pythium agar plate stored at 4℃, and use an inoculation needle to pick up an edge piece of about 5 mm × 5 mm and transfer it to a new semi-seawater corn agar medium. Place the plate in a constant temperature incubator at 24℃ for about 7 days for later use. 24℃ is the optimal growth temperature for Pythium porphyria.

[0057] The *Pythium sibiricum* strain used for initial screening was NBRC No. 33253. After igniting and cooling the punch with an alcohol lamp, 5 mm circular mycelial cakes were created at the edge of the *Pythium sibiricum*. These cakes were then transferred to new semi-seawater corn agar plates and incubated at 24℃ for 1 day. A cross was drawn with the *Pythium sibiricum* cake as the center as the positioning line. After igniting and cooling the inoculation loop with an alcohol lamp, a single colony of activated bacteria was picked and placed 2 cm from the center of the *Pythium sibiricum*. A short line of 1 cm was drawn perpendicular to the positioning line. Each plate could be inoculated with 4 bacterial strains. The control group was not inoculated with *Pythium sibiricum*. The plates were incubated at 24℃. When the diameter of the *Pythium sibiricum* in the control group reached approximately 4 cm, an inhibition zone was observed around the bacteria on the opposing plate. Strains showing antibacterial activity against *Pythium sibiricum* were selected, and the experiment was repeated. Each opposing plate was inoculated with the same strain, equivalent to 4 replicates. When the *Pythium sibiricum* in the control group reached 4 cm, the distance between the edge of the *Pythium sibiricum* on the positioning line and the bacteria was measured with calipers. Single colonies of the antagonistic bacteria were selected from the screening and inoculated into Zobell 2216E liquid medium. The colonies were then shaken overnight and stored in Zobell 2216E liquid medium containing 30% glycerol at -80°C.

[0058] like Figure 1As shown, through confrontation experiments, nine bacterial strains with antagonistic effects against the growth of *Pythium spp.* NBRC NO. 33253 were screened from 385 isolated and purified bacterial strains. The results of the differential significance analysis showed that, under 24℃ conditions, these nine strains antagonized *Pythium spp.* NBRC NO. 33253, with the inhibition zone size P19 > P3 > P6 > P8 > P4 > B1, P7, P12 > P5.

[0059] The *Pythium sibiricum* strain used for secondary screening was NBRC No. 33253, a strain that showed antagonistic activity against *Pythium sibiricum* in the initial screening. After igniting the inoculation loop with an alcohol lamp and cooling it, a single colony was picked and inoculated into 100 mL of Zobell 2216E liquid medium. The medium was incubated at 28°C and 150 r / min with shaking for 72 h. 30 mL of the bacterial culture was transferred to a 50 mL sterile centrifuge tube and centrifuged at 5000 r / min for 10 min at 4°C. The supernatant was filtered through a 0.22 μ nitrocellulose membrane to obtain a sterile filtrate for later use. The sterile semi-seawater corn agar medium was cooled to approximately 40°C, and the sterile filtrate was thoroughly mixed with the semi-seawater corn agar medium at a ratio of 1:5 before plating. After igniting and cooling the punch with an alcohol lamp, create 5 mm circular mycelial cakes from the edge of *Pythium sibiricum*. These cakes are then inoculated into the center of a plate containing mycelial solution as the experimental group. A strain that did not show antagonistic activity against *Pythium sibiricum* in the initial screening is selected and inoculated with *Pythium sibiricum* mycelial cakes as a negative control. *Pythium sibiricum* is inoculated onto semi-seawater corn agar plates without mycelial solution as a blank control. Each group of plates is prepared in triplicate and incubated at 24℃. Once the blank group of *Pythium sibiricum* has completely colonized the plate, all plates are photographed using a colony analyzer (Synbiosis Protocol 2, UK). The target area is circled using the "arbitrary polygon" tool on the AJ-VERT software of an OLYMPUS BX53 (Japan), and the unit length is measured using the "line segment" measuring tool. The inhibition rate is statistically analyzed and calculated using Excel software. The calculation formula is as follows:

[0060] Antibacterial rate = (Pythium area in control group - Pythium area in experimental group) / (Pythium area in control group - Initial Pythium area in inoculated group) × 100%

[0061] The re-screening experiment revealed that when the control group's *Pythium sibiricum* NBRC NO. 33253 completely covered the plates, *Pythium sibiricum* growing on plates containing sterile filtrate of negative-positive strains and antagonistic bacteria B1, P4, P5, P7, P8, and P12 also completely covered the plates. This indicates that the sterile filtrate of negative-positive strains and antagonistic bacteria B1, P4, P5, P7, P8, and P12 did not inhibit *Pythium sibiricum* growth. However, the growth of *Pythium sibiricum* was significantly inhibited on plates containing sterile filtrate of antagonistic bacteria P3, P6, and P19. The growth inhibition rates of *Pythium sibiricum* by the fermentation broth of antagonistic bacteria P3, P6, and P19 were 20.04%-21.51%, 21.56%-23.12%, and 27.66%-30.09%, respectively. The results of the significance analysis showed that the sterile filtrate of P19 had a significantly stronger inhibitory effect on Pythium growth than the sterile filtrates of P3 and P6, while there was no significant difference in the inhibitory effect of the sterile filtrates of P3 and P6 on Pythium growth. Based on the initial screening and rescreening results, it was considered that antagonistic strains P3, P6, and P19 had good antagonistic potential against Pythium latifolium, and therefore these three antagonistic bacteria were selected for further experiments.

[0062] Table 1. Statistical table of inhibition rate of antagonistic bacteria sterile filtrate on Pythium growth.

[0063]

[0064] Antagonistic spectrum of antagonistic bacteria against Pythium porphyria in Porphyra

[0065] Based on the results of the initial and secondary screening experiments, three antagonistic bacteria (P3, P6, and P19) with the best antagonistic effects were selected. Their antagonistic spectrum against eight laboratory-preserved *Pythium* strains was measured at 15℃ and 24℃ to evaluate the antagonistic ability of the three antagonistic bacteria against *Pythium* at the optimal growth temperature of 24℃ and the laboratory temperature for *Pythium* infection of *Porphyra* at 15℃. After the inoculation loop was ignited with an alcohol lamp and cooled, antagonistic bacteria were picked and inoculated into 50 mL of Zobell 2216E liquid medium. The culture was incubated at 28℃ and 150 r / min for 12 h with shaking. The bacterial concentration was adjusted to OD600 = 1.000 using Zobell 2216E liquid medium as a seed culture, and 10% of this seed culture was transferred to 100 mL of Zobell 2216E liquid medium. The culture was incubated at 28℃ and 150 r / min for 1-2 h with shaking. Take 30 mL of bacterial culture into a 50 mL sterile centrifuge tube, centrifuge at 5000 r / min for 10 min at 4℃, discard the supernatant, resuspend the bacterial cells in sterile seawater, and repeat the centrifugation and resuscitation steps 3 times to prepare 10 8A CFU / mL bacterial suspension was prepared. Using the confrontation method from section 2.3.2 (initial screening), the 1 cm short line was replaced with a 2 μL antagonistic bacterial suspension, with the same antagonistic bacterium spotted at four locations as the experimental group. A plate containing 2 μL of sterilized seawater was spotted 2 cm away from the Pythium cake as the blank control group. The plates were incubated at 15℃ and 24℃, with three replicates for each group. When the Pythium sibiricum in the control group completely covered the plate, all plates were photographed.

[0066] like Figure 2 As shown, under the optimal growth temperature of 24℃ for *Pythium sibiricum* and the laboratory infection temperature of 15℃, antagonistic bacteria P3, P6, and P19 all exhibited antagonistic effects on the growth of eight *Pythium* strains preserved in the laboratory. At 24℃, the inhibition rates of antagonistic bacteria P3 on the growth of the eight *Pythium* strains were 29.45%-96.27%, P6 was 28.78%-88.33%, and P19 was 91.11%-99.19%. At 15℃, the inhibition rates of antagonistic bacteria P3 on the growth of the eight *Pythium* strains were 52.09%-97.95%, P6 was 26.81%-78.04%, and P19 was 10.47%-41.91%.

[0067] Molecular identification of antagonistic bacteria

[0068] like Figure 3 As shown, BLAST homology alignment of the 16S rRNA sequencing results in GeneBank revealed that the 16S rRNA sequences of P3, P6, and P19 showed the highest similarity to *Pseudoalteromonas* sp., with P3 and P6 showing 99.93% similarity and P19 showing 99.86% similarity. Multilocus sequence analysis enabled more accurate classification and identification of the antagonistic bacteria P3, P6, and P19. Phylogenetic analysis revealed that in the phylogenetic tree constructed based on the four genes 16S rRNA, dnaA, dnaN, and recA, antagonistic bacteria P3 and P6 clustered with *Pseudoalteromonas piscicida*, while antagonistic bacteria P19 clustered with *Pseudoalteromonaspeptidolytica*.

[0069] The confrontational growth of Pythium and antagonistic bacteria

[0070] like Figure 4 and Figure 5Antibacterial spectrum determination showed that antagonistic bacteria P3, P6, and P19 all exhibited antagonistic activity against the eight Pythium strains preserved in our laboratory. Among them, Pythium 00, 26, 33, and 53 were Pythium porphyare, and Pythium JS, HT, LS, and RZ were Pythium chondricola. This indicates that the three antagonistic bacteria screened out have antagonistic activity against both Pythium porphyare and Pythium chondricola. Analysis of significant differences showed that antagonistic bacterium P3 exhibited enhanced inhibitory effects against Pythium 00, 26, JS, and LS at 15℃ compared to 24℃, while its inhibitory effects against Pythium 33, 53, HT, and RZ showed no significant difference between the two temperature conditions. Antagonistic bacterium P6 showed enhanced inhibitory effects against Pythium 00 at 15℃ compared to 24℃, and its inhibitory effects against Pythium 53, JS, HT, and RZ were significantly stronger at 24℃ than at 15℃. However, its inhibitory effects against Pythium 26, 33, and LS showed no significant difference between the two temperature conditions. Antagonistic bacterium P19 showed significantly reduced inhibitory effects against all eight Pythium strains at 15℃ compared to 24℃. These findings indicate that temperature had a certain impact on the antagonistic activity of the three antagonistic bacteriums. Because the antagonistic effect of P19 against the eight strains of Pythium significantly decreased at 15℃, and 15℃ is the optimal temperature for Pythium infection of laver under laboratory conditions, it was deemed unsuitable as a potential strain for antagonizing laver red rot disease and was therefore excluded from subsequent experiments. Based on this experiment, antagonistic bacteria P3 and P6 were selected as potential strains for controlling red rot disease.

[0071] Safety evaluation of antagonistic bacteria on laver

[0072] Take 10 mL of bacteria with a concentration of 10 8 A suspension of antagonistic bacteria at CFU / mL was placed in a 250 mL Erlenmeyer flask, and 90 mL of sterile seawater was added to bring the final concentration of the antagonistic bacteria in the flask to 10. 7 CFU / mL, each bottle contained 0.2 g of laver as the experimental group; 0.2 g of laver was added to 100 mL of sterilized seawater as the control group. Each group had three replicates. Culture conditions were: static culture, temperature 15℃, light intensity 62.5 μmol photons / m². -2 s -1 The photoperiod was L:D = 12:12, with PES added every 3 days, and the water was not changed. After sampling on day 12 of the experiment, each group of laver was rinsed three times in sterilized seawater and then transferred to 500 mL aeration bottles containing sterilized seawater for aeration cultivation. The cultivation conditions were: aeration cultivation in aeration bottles with aeration tubes, temperature 15℃, and light intensity 62.5 μmol photons / m². -2 s -1The photoperiod was L:D = 12:12, and the water was changed and PES was added every 3 days. The experiment was stopped on day 18.

[0073] like Figure 6 As shown, the activity of immune defense enzymes in each group of laver was measured every 3 days. ROS activity assays showed that throughout the experiment, the ROS activity of laver soaked in antagonistic bacterial suspensions (P3 and P6 at a concentration of 10⁷ CFU / mL) was not significantly different from that of the control group. MAD activity assays showed that the MDA activity of laver soaked in P3 bacterial suspension at a concentration of 10⁷ CFU / mL was significantly higher than that of the control group at 3 and 6 days. On day 9 of the experiment, the MDA activity of laver recovered to the same level as the control group, and thereafter, there was no significant difference from the control group until the end of the experiment. 7 On day 3 after soaking in P6 bacterial suspension at CFU / mL, the MDA activity of the seaweed was significantly higher than that of the control group. On day 6 of the experiment, the MDA activity of the seaweed recovered to the same level as the control group, and thereafter there was no significant difference from the control group until the end of the experiment.

[0074] like Figure 7 As shown, chlorophyll fluorescence parameters were measured every 3 days using a FluorCam closed-system chlorophyll fluorescence imaging system (FluorCam 800MF, Czech PSI). The results of the maximum photochemical quantum yield Fv / Fm and the photochemical quantum yield φPSII showed that after transferring the laver soaked in bacterial solution for 12 days to normal water for 6 days of cultivation, there was no significant difference between the experimental group and the control group.

[0075] like Figures 8-9 As shown, the macroscopic morphology and microscopic morphology of each group of laver were observed every 3 days. The results of the laver growth rate measurement showed that ( Figure 8 During the seven sampling observations throughout the experiment, the bacterial concentration was 10... 7 The length of *Porphyra* grown in P3 or P6 bacterial suspensions at CFU / mL showed no significant difference compared to the control group. At the end of the experiment, the bacterial concentration was 10... 7 No visible lesions appeared in the seaweed soaked in CFU / mL P3 or P6 bacterial suspension for 12 days, and under a microscope, the seaweed cells in the bacterial suspension soaking group showed no obvious abnormalities compared with the control group.

[0076] In summary, the antagonistic bacteria at this concentration in the method described in this invention will not cause rot or decay in laver, will not affect the cell morphology of laver, and will not inhibit laver growth. Therefore, it is considered that antagonistic bacteria P3 and P6 are not pathogenic strains of laver, and the bacterial concentration is 10... 7 CFU / mL of P3 or P6 bacterial suspensions can be used to investigate the antagonism of red rot disease in laver.

[0077] The inhibitory effect of antagonistic bacteria on red rot disease at different stages

[0078] Antagonistic bacteria pre-occupation experiment. In the experimental group, an antagonistic bacterial suspension was added to the *Porphyra yezoensis* culture system on day 1 of the experiment, with a final concentration of 10... 7 CFU / mL, on day 4, a suspension of Pythium zoospores was added to bring the final concentration of Pythium zoospores in the culture system to 10. 5 spores / mL; the positive control group was a Porphyra culture system in which Pythium zoospores were added alone on day 4, with a final concentration of Pythium zoospores of 10⁻⁶. 5 spores / mL; the blank control was a Porphyra culture system without the addition of Pythium zoospores and antagonistic bacteria; additionally, a final concentration of 10 was added on day 1. 7 The CFU / mL antagonistic bacteria culture system in Porphyra served as a control.

[0079] Pre-occupation experiment with Pythium. The experimental group consisted of a Porphyra culture system in which antagonistic bacteria were added on day 1 (day 2 of the experiment) or day 3 (day 4 of the experiment) after infection by Pythium zoospores. The final concentration of Pythium zoospores in the culture system was 10. 5 spores / mL, final concentration of antagonistic bacteria was 10 7 CFU / mL; the positive control group and blank control group were the same as in Scheme 1; additionally, on day 2 and day 4 of the experiment, a final concentration of 10 CFU / mL was added. 7 The CFU / mL antagonistic bacteria culture system in *Porphyra* served as a control. Specific groupings are shown in the table below:

[0080] Table 2 Experimental setup for antagonistic bacteria pre-occupation

[0081]

[0082] Table 3 Experimental setup for Pythium pre-occupation

[0083]

[0084] The results of the experiment on the resistance of antagonistic bacteria to Pythium infection are shown in the table below:

[0085] Table 4. Antagonistic bacteria pre-occupation experiment

[0086]

[0087] Table 5. Pre-occupation experiment of Pythium

[0088]

[0089] like Figure 10As shown, the antagonistic bacteria's effect on Pythium infection was observed visually. In summary, antagonistic bacteria P6 showed good control potential against red rot, therefore, antagonistic bacteria P6 was selected for the next stage of the experiment. The experimental results showed that when Pythium preferentially occupied the soil before treating laver with antagonistic bacteria P6, the lesion rate was significantly lower than that of laver infected with Pythium after antagonistic bacteria P6 preferentially occupied the soil.

[0090] Exploring the application methods of antagonistic bacteria P6

[0091] On day 1 of the experiment, laver was infected with zoospores of *Pythium spp.* NBRC 33253. The final concentration of zoospores in the laver culture system was 10. 5 spores / mL; On day 2 of the experiment, when Pythium mycelium was observed to invade the Porphyra cells, the Porphyra was removed from the culture water and allowed to dry for about 30 seconds until it stopped dripping. Then it was placed in a solution with a concentration of 10 7 Immerse the cells in a CFU / mL P6 bacterial suspension for different times (3 s, 30 s, 1 min, 3 min), then remove and spread them evenly in a 90 mm sterile petri dish. Place the petri dish vertically and incubate at 15℃ and an optical density of 62.5 μmol photonsm. -2 s -1 After drying for 1 hour under the specified conditions (water loss rate approximately 25%), the laver was rinsed three times with sterilized seawater and then returned to the sterilized seawater for further cultivation. On the fourth day of the experiment, pinhead-sized lesions were observed on the laver, and the laver was treated using the same method as on the second day. The above laver culture system served as the experimental group. Eight control groups were set up: laver culture system infected with Pythium but not treated with P6 and dried; laver culture system infected with Pythium but not treated with P6 and not dried; laver culture system not infected with Pythium but treated with antagonistic bacteria (3 s, 30 s, 1 min, 3 min) and dried; laver culture system not infected with Pythium, not treated with antagonistic bacteria but dried; and laver culture system not infected with Pythium, not treated with antagonistic bacteria and not dried. The specific tissue divisions are shown in the table below:

[0092] Table 6 Experimental setup for investigating the application of antagonistic bacteria P6 against red rot disease in laver.

[0093]

[0094] The results of the investigation into the application methods of antagonistic bacteria P6 are shown in the table below:

[0095] Table 7. Statistical table of lesion incidence and lesion inhibition rate of laver in each infection group.

[0096]

[0097] like Figure 11As shown, the antagonistic bacterium P6's effect on Pythium infection was observed with the naked eye.

[0098] Experimental results showed that the groups that were not subjected to Pythium infection: dried for only 1 hour and 10 hours... 7 No rot was observed in the laver groups treated with CFU / mL antagonistic bacteria P6 immersion (3s, 30s, 1min, 3min) followed by 1h drying. This indicates that neither the 1h drying nor the 107 CFU / mL antagonistic bacteria P6 immersion (3s, 30s, 1min, 3min) followed by 1h drying treatments will cause laver rot and can both be used for the control of laver red rot. Statistical results of laver lesion rates in each Pythium-infected group showed no significant difference in lesion rates between the laver group treated with 1h drying and the group not treated with drying, indicating that 1h drying alone has no significant inhibitory effect on red rot.

[0099] Use 10... 7 Soaking in a suspension of the antagonistic bacterium P6 at CFU / mL for 3 s, 30 s, 1 min, and 3 min followed by drying for 1 h all showed some inhibitory effect on red rot, with lesion inhibition rates of 40.91%-44.58%, 41.95%-61.84%, 52.54%-61.92%, and 77.23%-86.51%, respectively. Among these, 10 7 The lesion rate of laver in the group soaked in a suspension of antagonistic bacteria P6 for 3 min and then dried for 1 h was significantly lower than that in the other experimental groups, indicating that this method has the best inhibitory effect on red rot and has good potential for the prevention and control of red rot.

[0100] The specific embodiments described above are preferred embodiments of the present invention and are not intended to limit the specific scope of the present invention. The scope of the present invention includes, but is not limited to, these specific embodiments. All equivalent changes made in accordance with the present invention are within the protection scope of the present invention.

Claims

1. A microbial inoculant for controlling red rot disease in laver, characterized in that, It includes Pseudoalteromonas piscicida strain P3, strain P6 or their fermentation broth.

2. The microbial agent for controlling red rot disease of laver according to claim 1, characterized in that, The strain P3 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC No. M20221357; the strain P6 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC No. M20221356.

3. The microbial agent for controlling red rot disease of laver according to claim 1, characterized in that, The fermentation broth is prepared as follows: a. Prepare the fermentation medium, the components of which are as follows: Peptone 5.0 g / L, yeast extract 1.0 g / L, ferric citrate 0.1 g / L, sodium chloride 19.45 g / L, magnesium chloride 5.9 g / L, magnesium sulfate 3.24 g / L, calcium chloride 1.8 g / L, potassium chloride 0.55 g / L, sodium bicarbonate 0.16 g / L, potassium bromide 0.08 g / L, trace amounts of strontium salts and borates, prepared with aged seawater or artificial seawater, pH adjusted to 7.6-7.8, and autoclaved at 121℃ for 20 min; b. Slant activation: Inoculate the antagonistic bacteria P3 or P6, which are stored in glycerol tubes at -80℃, onto 2216E agar plates and incubate at 28℃ for 24-48 h. Select single colonies with neat edges and full shape. c. Seed culture: The activated single colony was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of 2216E liquid medium and cultured with shaking at 28℃ and 150 r / min for 12-18 h until the bacterial concentration reached OD600 of 1.0-1.2 to obtain the first-grade seed culture; d. Fermentation process in fermenter: Inoculate the above seed liquid into the fermenter at an inoculation rate of 5%-8% by volume; e. Whole-culture fermentation broth: The mixture after fermentation is complete is the whole-culture fermentation broth, at which point the bacterial concentration should reach 10. 9 -10 10 CFU / mL.

4. A method for preventing and controlling red rot disease in laver, characterized in that, The seaweed is soaked in a microbial preparation containing any one of claims 1-3.

5. The method for preventing and controlling red rot disease in laver according to claim 4, characterized in that, The concentration of *Pseudomonas aeruginosa* in the microbial preparation is 10. 6 -10 8 CFU / mL.

6. The method for preventing and controlling red rot disease in laver according to claim 5, characterized in that, The method also includes drying the laver after soaking.

7. The method for preventing and controlling red rot disease of laver according to claim 6, characterized in that, The soaking time is 3 seconds to 3 minutes; the drying time is 0.5 hours to 1.5 hours.

8. The method for preventing and controlling red rot disease of laver according to claim 7, characterized in that, The method involves using a concentration of 10. 7 The seaweed was soaked in a solution of strain P6 with CFU / mL for 3 minutes, and then dried for 1 hour.

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