Koreanibacterium sp. lm inhibiting saprolegnia and its use

Abstract

The present application relates to the field of microbial technology, and more particularly to a Pseudomonas koreensis LM for inhibiting fish pathogenic fungus Saprolegnia and application thereof.The Pseudomonas koreensis LM is classified and named as Pseudomonas koreensis , and was preserved in China Center for Type Culture Collection on November 15, 2019, with a preservation number of CCTCC NO: M 2019934.The Pseudomonas koreensis LM provided in the present application has good inhibitory activity on mycelium growth and spore germination of fish pathogenic fungus Saprolegnia, and has stable bacteriostatic activity, good biological safety, and potential value for development as a green and environmentally friendly Saprolegnia biological prevention and control agent.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a strain of Korean Pseudomonas LM that inhibits the fish pathogenic fungus Saprolegnia and its applications. Background Technology

[0002] Water mold ( Saprolegnia Saprolegniasis is a widely distributed opportunistic pathogenic fungus in aquatic bodies, infecting freshwater fish eggs, fry, and adult fish. It spreads through zoospores. Due to its lack of strict host selectivity, ability to produce biofilm resistance, and secretion of effector proteins that interfere with host immunity, the control of Saprolegniasis has become one of the most challenging fish diseases. Malachite green, a previously effective drug for Saprolegniasis, was banned worldwide in 1992 due to its carcinogenic, teratogenic, and mutagenic effects. However, due to the lack of effective drugs for Saprolegniasis control, malachite green remains a persistent problem, posing a significant threat to the quality and safety of aquatic products. Developing highly effective and safe alternatives to malachite green to ensure the quality of aquatic products and protect the aquatic ecosystem has become an urgent priority.

[0003] Biological antagonism-based methods for the prevention and control of aquatic animal diseases have become a research hotspot in the aquaculture field due to their green and environmentally friendly nature. Among the genera of bacteria reported to have antagonistic effects against Saprolegnia are: Aeromonas intermedius (…). Aeromonas media Serratia marcescens ( ) Serratia marcescens ), Bacillus spp. ( Bacillus Streptomyces ( Streptomyces )wait.

[0004] The optimal temperature range for the growth and reproduction of Saprolegnia is 5-26 ℃. Most existing Saprolegnia antagonistic bacteria are isolated and screened from soil and aquaculture water environments, and their optimal growth temperature is relatively high. Therefore, they cannot achieve the best antibacterial effect within the optimal temperature range for the growth and reproduction of Saprolegnia. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a strain of Pseudomonas koreanum LM that inhibits the pathogenic fungus Saprolegnia in fish and its applications.

[0006] One of the objectives of this invention is to provide a strain of *Pseudomonas koreanum* that inhibits the pathogenic fungus *Saprolegnia* in fish. Pseudomonas koreensis LM was deposited at the China Center for Type Culture Collection on November 15, 2019, with accession number CCTCC NO: M 2019934.

[0007] The Korean Pseudomonas (Korean Pseudomonas) strain obtained by the first screening in this invention Pseudomonas koreensis LM has an inhibitory effect on the fish pathogenic fungus Saprolegnia, and its anti-Saprolegnia activity is genetically stable, acid and alkali stable, non-pathogenic to mammals and fish, and has good biosafety. It can be used as a candidate strain for the development of biocontrol agents for fish pathogenic fungi.

[0008] The currently reported Saprolegnia antagonists, such as Bacillus, Streptomyces and the like, are mostly obtained by screening from soil and aquaculture water environment, and a new Saprolegnia antagonistic bacterium is successfully isolated and screened from Saprolegnia epiphyte in the application. The growth and reproduction of Saprolegnia is suitable for the temperature range of 5-26 ℃, and the Korean Pseudomonas LM of the application can grow at 4 ℃, and still has good Saprolegnia inhibitory activity at 15 ℃ low temperature, which provides a scientific basis for the direct application of LM to the prevention and control of Saprolegnia infection and transmission in aquaculture water.

[0009] The Saprolegnia infection test results of the LM bacteria on sticky fish eggs show that the LM bacteria have better Saprolegnia infection resistance effect than the positive control (MG) of malachite green, and are expected to replace malachite green for the prevention and control of Saprolegnia disease in artificial hatching of sticky fish eggs, and reduce water drug residues.

[0010] The second purpose of the application is to provide a bacterial agent, wherein the active ingredient of the bacterial agent comprises the Korean Pseudomonas LM or the fermentation liquor obtained by fermenting the Korean Pseudomonas LM.

[0011] The third purpose of the application is to provide the application of the Korean Pseudomonas LM or the bacterial agent in aquaculture of aquatic animals.

[0012] Further, the Korean Pseudomonas LM or the bacterial agent is prepared into a drug for resisting pathogenic bacteria of aquatic animals.

[0013] Further, the aquatic animals include fish.

[0014] Further, the fish is freshwater fish.

[0015] Further, the pathogenic bacteria is fungus.

[0016] Further, the fungus includes Saprolegnia.

[0017] The fourth purpose of the application is to provide a drug, wherein the drug comprises the Korean Pseudomonas LM or the bacterial agent.

[0018] Further, the drug further comprises pharmaceutically acceptable adjuvants, such as diluents (water), freeze-drying protectants (mannitol, trehalose, lactose, sucrose, glycerol, amino acids), pH regulators (sodium bicarbonate, citric acid).

[0019] The application has the following beneficial effects:

[0020] The Pseudomonas koreensis LM of the application has genetic stability of inhibiting Saprolegnia activity, and is stable to acid and alkali; the inhibition rate of the Pseudomonas koreensis LM on Saprolegnia mycelium growth is about 70%, and the diameter of the inhibition zone of the Pseudomonas koreensis LM on Saprolegnia spore germination is more than 30 mm; the Pseudomonas koreensis LM has no infection effect on the skin of mammals and no pathogenicity to fish; the Saprolegnia infection rate of the test group treated by the Pseudomonas koreensis LM with a concentration of 2.0×10 7 The Saprolegnia infection rate of the test group treated by the Pseudomonas koreensis LM with a concentration of 2.0×10 BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 A: Saprolegnia and epiphyte bacteria from green crucian carp; B: Saprolegnia and epiphyte bacteria from mandarin fish; C: purified epiphyte bacteria JM from green crucian carp; D: purified epiphyte bacteria LM from mandarin fish; E: purified Saprolegnia pathogenic bacteria JM19.

[0022] Figure 2 A: the inhibition effect of epiphyte bacteria LM and JM from different sources on Saprolegnia pathogenic bacteria JM19 at 15℃; B: the inhibition effect of epiphyte bacteria LM and JM from different sources on Saprolegnia pathogenic bacteria JM19 at 25℃.

[0023] Figure 3 A: the inhibition effect of LM bacteria on Saprolegnia at different generations (CK: Saprolegnia pathogenic bacteria JM19 blank control; G1, G5 and G10 represent the first generation, the fifth generation and the tenth generation of LM bacteria, respectively); B: comparison of the inhibition activity of LM bacteria on Saprolegnia at different generations (G1, G5 and G10 represent the first generation, the fifth generation and the tenth generation of LM bacteria, respectively); a: the same letter indicates that there is no significant difference in the inhibition activity of each generation, P>0.05.

[0024] Figure 4The results of the acid-base stability detection of the Saprolegnia-inhibiting activity of the LM bacteria are shown in A and B, and the comparison of the Saprolegnia-inhibiting activity of the LM bacteria after different pH treatments is shown in C and D (a: same letters mean that there is no significant difference in the Saprolegnia-inhibiting activity of the LM bacteria under different pH conditions, P>0.05).

[0025] Figure 5 The results of the heat stability detection of the Saprolegnia-inhibiting activity of the LM bacteria are shown in A and B, and the comparison of the Saprolegnia-inhibiting activity of the LM bacteria after different temperature treatments is shown in C and D (a, b: t-test results of the inhibition rate of each group, same letters mean that there is no significant difference, P>0.05, and different letters mean that there is a significant difference, P<0.05).

[0026] Figure 6 The results of the mammalian skin sensitivity test of the LM bacteria are shown in A and B.

[0027] Figure 7 The results of the morphological observation of the LM bacteria are shown in A and B.

[0028] Figure 8 The results of the molecular biology identification of the LM bacteria are shown in A and B.

[0029] Figure 9 The test diagram of the effect of the cut Xiangyun carp artificial insemination egg mesh on the Saprolegnia infection of the LM bacteria is shown in A and B. DETAILED DESCRIPTION

[0030] The present application will be described in detail below in conjunction with the accompanying drawings and specific examples, but should not be understood as a limitation of the present application. If not specifically stated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, if not specifically stated, can be obtained from commercial channels.

[0031] Example 1: Isolation and identification of water mold bacteria.

[0032] 1. Test method

[0033] 1.1 Sample collection

[0034] On March 15, 2019, one live Squalidus argentatus infected with mild water mold and three Siniperca chuatsi infected with severe water mold and died shortly were collected from the on-campus aquatic training base of Hunan University of Science and Technology. The water mold on the parasitic Squalidus argentatus was located on the abdomen between the left and right pectoral fins of the fish body, with white and old cotton-like mycelium up to 2 cm long. The infection area of water mold on the body surface of Siniperca chuatsi was more than 80%, with shorter mycelium and brownish yellow color.

[0035] 1.2 Preparation of culture medium

[0036] LB medium: tryptone (1%), yeast extract (0.5%), NaCl (1%), agar powder (1.8%, added when preparing solid medium); PDA plate: potato (20%), glucose (2%), agar powder (2%); test tube slant: the formula is the same as that of PDA plate.

[0037] 1.3 Isolation and purification of water mold pathogen and its epiphytic bacteria

[0038] On the clean bench, the mycelium on the surface of Squalidus argentatus and Siniperca chuatsi infected with water mold was repeatedly washed with sterile water for 4-5 times, and the water mold was inoculated into the center of the PDA plate with an inoculation needle. After 48 h of constant temperature culture at 25℃, it was observed that the water mold mycelium from Squalidus argentatus and Siniperca chuatsi grew radially from the center to the edge of the PDA plate, but with the extension of culture time, the outer mycelium appeared water death phenomenon, and a large number of epiphytic bacteria were found in the water mold mycelium by microscopic examination. The purification of pathogenic fungus water mold was carried out by agar block transfer method. Fresh water mold blocks were punched and removed from the edge of the strain separation plate before water mold hydration, and transferred to the center of a new PDA plate for low-temperature culture at 15℃. After repeated transfer for 2-3 times, pure water mold was obtained, which was transferred to a PDA plate for constant temperature culture at 25℃ for 5 days, and stored in a 4℃ refrigerator for standby. The isolation and purification of epiphytic bacteria were carried out by streaking separation method. The bacteria were inoculated into LB plate from the center of PDA culture plate, and purified by repeated streaking for 2-3 times. The purified strain was transferred to PDA test tube slant, and stored in a 4℃ refrigerator for standby.

[0039] 1.4 Screening of water mold inhibiting bacteria

[0040] The isolated and purified water mold pathogen was used as the test strain, and the agar block transfer method was used to transfer the water mold block to the center of the PDA plate. The purified epiphytic bacteria were spotted 1 cm away from the water mold block, and the confrontation growth was carried out at 15℃ and 25℃ for 4 days. The bacteria with obvious water mold growth inhibition effect were selected and named LM, which is referred to as LM bacteria hereinafter.

[0041] 1.5 Stability detection of LM bacteria antibacterial activity

[0042] Genetic stability detection: LM bacteria were continuously subcultured on PDA slant for 10 generations. The 1st, 5th and 10th generations were used for detection of genetic stability of antibacterial activity by Oxford cup method. Saprolegnia bacteria were inoculated on PDA plates by agar block method, and 4 Oxford cups were placed at equal intervals about 2 cm from the center, with 80 μL of fermented liquid of LM bacteria of different generations (25°C, 120 r / min shaking culture for 24 h) added into each cup. After 48 h of constant temperature culture at 25°C, the growth diameter of Saprolegnia was observed and measured, with the blank culture liquid as control. The inhibition rate was calculated, with 3 parallel groups in each group. Inhibition rate = (diameter of Saprolegnia in control group - diameter of Saprolegnia in test group) / diameter of Saprolegnia in control group.

[0043] Thermal stability detection: LM bacteria fermented liquid was treated in water bath at 40°C, 60°C, 80°C and 100°C for 1 h, respectively. The thermal stability of Saprolegnia mycelium growth and spore germination was detected by Oxford cup method, with inhibition rate and inhibition zone size as indexes.

[0044] Acid-base stability detection: The original pH value of LM bacteria fermented liquid was 5.0, which was adjusted to 7.0 and 9.0 by 1 mol / L NaOH solution. After 1 h of room temperature standing, the samples treated with alkali were adjusted back to the original pH value of the fermented liquid by 1 mol / L HCl solution. The activity of different treated samples in inhibiting Saprolegnia mycelium growth and spore germination was detected by Oxford cup method.

[0045] 1.6 Biological safety detection

[0046] Skin sensitivity test: SPF mice with a body weight of 18-22 g (purchased from Hunan Slike Jingda Experimental Animal Co., Ltd.) were used as test objects, with two males and two females in each group, which were divided into two groups (test group and control group), with one male and one female in each group. The mice in the test group were fixed on a wooden board with rubber bands, and the fur on their abdominal skin was removed. LM bacteria were evenly coated on the exposed skin with a cotton swab. After 20 min of immersion, the mice were released to move freely. The fur on the abdomen of the mice in the control group was also removed, and they were released after 20 min. The mice in the test and control groups were raised separately. Special maintenance feed was fed to the mice, and they were free to drink water. The experimental site, cage and water were cleaned and disinfected on time. The skin condition of the mice was observed and photographed for 3 days.

[0047] Fish challenge experiment: Crucian carp fry with an average body length of 8.5 cm and a weight of 6.8 g were randomly divided into four groups (blank control group, PBS injection group, LM immersion group, and LM injection group), with 10 fish in each group. Each fish in the PBS injection group received an intrathoracic injection of 0.1 mL of PBS, and each fish in the LM injection group received an injection of 0.1 mL of PBS at a bacterial concentration of 1×10⁻⁶. 8 The bacterial solution was prepared at cfu / mL, while the LM immersion group had the bacterial solution added directly to the basin to achieve an LM bacterial concentration of 1.0 × 10⁻⁶. 7 cfu / mL. Observe for 16 days, without feeding, and add an appropriate amount of fresh water every 2 days. Observe and record the activity and survival of the fish.

[0048] 1.7 Species Identification

[0049] LM bacteria were cultured on LB agar plates to observe colony morphology; Gram staining was used for microscopic observation of bacterial cell morphology; and microbiological identification was performed using bacterial micro-biochemical identification tubes (purchased from Hangzhou Microbial Reagent Co., Ltd.). Molecular biological identification was performed using 16S rDNA sequence analysis. The specific procedure was as follows: using LM genomic DNA as a template and 27F / 1492R as primers, 16S rDNA was amplified by PCR. The PCR reaction system (50 µL) consisted of: ddH2O 20.0 µL, 27F 2.0 µL, 1492R 2.0 µL, DNA template 1.0 µL, and 2×Taq PCR Master Mix 25.0 µL. The reaction program was: first 94 ℃ for 5 min, then 94 ℃ for 30 s, 58 ℃ for 45 s, 72 ℃ for 2 min, for 30 cycles, and finally 72 ℃ for 10 min. The PCR products were purified by gel extraction and sent to Shanghai Bioengineering Co., Ltd. for sequencing. The sequences were aligned using BLAST on the NCBI website, and a phylogenetic tree was constructed using MEGA 5.2 software.

[0050] 2. Test Results

[0051] 2.1 Results of isolation and purification of Saprolegnia pathogen and its epiphytic bacteria

[0052] Saprolegnia cucumeroides grows radially from the center to the edge on PDA plates. As the culture time increases, the outer hyphae of Saprolegnia cucumeroides become hydrated and die, turning into a paste-like substance. Figure 1 A), microscopic examination revealed epiphytic bacteria within the Saprolegnia hyphae. Similar phenomena were observed in the Saprolegnia hyphae of mandarin fish on PDA plates; furthermore, the epiphytic bacteria produced a brownish-yellow pigment (A). Figure 1 B). Further isolation and purification yielded two epiphytic bacteria, JM ( Figure 1 C) and LM Figure 1 D), and a strain of Saprolegnia pathogen JM19 ( Figure 1 E).

[0053] 2.2 Screening results of water-suppressing mold bacteria

[0054] Using the pathogenic fungus JM19 as the test subject, it was cultured against epiphytic bacteria JM and LM at 15℃ and 25℃ for 48 h. The results showed that LM exhibited better inhibitory activity against water mold under both 15℃ and 25℃ conditions, producing a clear zone around the bacteria. The inhibitory activity was even higher at 25℃, while epiphytic bacteria JM did not produce any inhibitory effect against water mold. Figure 2 ).

[0055] 2.3 Results of the stability test of LM bacteria's antifungal activity against water mold

[0056] Using Saprolegnia glutinosa JM19 as the target bacterium, the genetic stability, acid-base stability, and heat stability of the antibacterial activity of LM bacteria were detected using the Oxford cup method. The results are shown in [Figure number missing]. Figures 3-5 .

[0057] Depend on Figure 3 It can be seen that the fermentation broths of the 1st, 5th, and 10th generations of LM bacteria have a significant inhibitory effect on the tested Saprolegnia pathogen. Figure 3 A) The inhibition rate of LM bacteria on the growth of Saprolegnia mycelium was 60%~70% in all generations, and there was no significant difference in inhibition rate between generations (P>0.05). Figure 3 B) indicates that the genetic stability of the LM bacteria's activity against water mold is good.

[0058] Depend on Figure 4 It can be seen that the fermentation broth of LM bacteria has a significant inhibitory effect on the growth of Saprolegnia mycelium under pH conditions of 5, 7, and 9. Figure 4 A) The antibacterial rate reached over 65%, and there was no significant difference in antibacterial rate under different pH conditions (P > 0.05). Figure 4 C). LM bacteria also showed a significant inhibitory effect on the germination of Saprolegnia spores under pH conditions of 5, 7, and 9. Figure 4 B), the diameter of the inhibition zone was greater than 32 mm, and there was no significant difference in the size of the inhibition zone under different pH conditions (P>0.05). Figure 4 D). This indicates that the inhibitory activity of LM bacteria against water mold is stable against acid and alkali conditions.

[0059] Depend on Figure 5 It is evident that the LM bacteria fermentation broth, after heat treatment at 40℃, still exhibits good antagonistic activity against the mycelial growth of Saprolegnia pathogens. Figure 5 A), with an inhibition rate of 71.3%, the antibacterial activity of the fermentation broth was not significantly different from that of the control group fermentation broth (cultured at 25℃) (P>0.05). After heat treatment at temperatures above 60℃, LM bacteria lost their activity in inhibiting the growth of Saprolegnia mycelium. Figure 5C); LM fermentation broth after heat treatment at 40℃ also had obvious inhibitory effect on the spore germination of S. striatum Figure 5 B), the average diameter of the inhibition zone reached 31.33 mm, and there was no significant difference compared with the control group (P>0.05), but the antagonistic activity of LM bacteria treated at a temperature above 60℃ on the spore germination of S. striatum was completely lost, and there was a significant difference compared with the control (P<0.05, Figure 5 D). This shows that the activity of LM bacteria in inhibiting S. striatum can be stable below 40℃.

[0060] 2.4 Results of biological safety test of LM bacteria

[0061] In the skin sensitivity test, the mice in the test group and the control group did not show symptoms of skin redness, ulceration and infection, and compared with the control, the mice in the test group had no obvious special symptoms, indicating that LM bacteria had no infection effect on the skin of mammals. Figure 6 ).

[0062] Fish challenge test showed that whether LM bacteria were soaked or injected, there was no difference in the activity and survival of green crucian carp compared with the control group (Table 1), indicating that LM bacteria had no pathogenicity to fish.

[0063] Table 1 Results of LM bacteria challenge test on green crucian carp

[0064]

[0065] Note: PBS: 0.7% phosphate balanced saline.

[0066] 2.5 Results of LM bacteria species identification

[0067] The LM bacterial colony morphology was round, brownish yellow, with a neat edge and a smooth and moist surface; it was a gram-negative bacillus Figure 7 ); physiological and biochemical identification results showed that LM bacteria could utilize glucose, xylose, urea, β-galactoside, esculin, ornithine decarboxylase, lysine decarboxylase, arginine decarboxylase, had hydrogen peroxide and oxidase, and could grow at 4℃ low temperature, but not at 37℃ (Table 2), which was basically consistent with the characteristics of Pseudomonas in the "Berger's Systematic Bacteriology Identification Manual".

[0068] Table 2 Physiological and biochemical identification results of LM bacteria

[0069]

[0070] Note: +: positive test result, -: negative test result, ±: weak positive test result.

[0071] 16S rDNA gene sequence (shown in SEQ ID NO. 1) analysis showed that LM bacteria were closely related to Pseudomonas koreensisstrain Ps 9-14 had a similarity of up to 98.88%, and clustered into a cluster on the phylogenetic tree Figure 8 ), the closest relationship, combined with its morphological, physiological and biochemical characteristics, it was identified as Pseudomonas koreensis ( Pseudomonas koreensis ), named LM, and preserved in China Center for Type Culture Collection on November 15, 2019, the preservation address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M 2019934.

[0072]

[0073] Example 2: Anti-A. hydrophila infection test of sticky fish eggs treated with different concentrations of LM bacteria.

[0074] 1. Test method

[0075] The uniformly distributed Xiangyun carp artificial insemination fish egg mesh was used as the test material (donated by Yang Futong, Technical General Engineer of Xiangyun Biotechnology Co., Ltd. Changde Base), which was cut into small square mesh with a side length of about 6 cm, and then inserted into the hatching cup (upper diameter 7.5 cm, bottom diameter 5.5 cm, height 7 cm) after counting the total number of eggs and fertilized eggs. Figure 9 The hatching cup was added with an appropriate amount of LM bacteria fermentation broth (25 ℃, 120 r / min shaking culture for 40 h), and 2.0×10 4 cfu / mL, 2.0×10 5 cfu / mL, 2.0×10 6 cfu / mL, 2.0×10 7 cfu / mL were set as four LM bacteria concentration treatment test groups, and the hatching cup without adding LM bacteria was set as the control group (CK group), and the hatching cup with a concentration of 0.1 g / m 3 Malachite green hatching cup was set as the positive control group (MG group), and each group had 3 replicates. The hatching cups were placed in a constant temperature incubator at 25 ℃ for 48 h, and then the number of A. hydrophila infected fish eggs was counted, and the number of fish fry in each cup was counted after hatching for 1-2 d until all the fish fry were hatched. The A. hydrophila infection rate and the fish fry hatching rate of each group were calculated according to the following formula.

[0076] (1) Fish egg A. hydrophila infection rate = number of A. hydrophila infected fish eggs on the mesh / total number of fish eggs on the mesh.

[0077] (2) Fish fry hatching rate = number of hatched fish fry in the hatching cup / number of fertilized eggs on the mesh in the cup.

[0078] 2. Test results

[0079] The effects of sticky fish eggs treated with different concentrations of LM bacteria on their resistance to A. hydrophila infection are shown in Table 3. Compared with the control group (CK) without adding LM bacteria, the four LM bacteria concentration treatment test groups significantly reduced the A. hydrophila infection rate of Xiangyun carp fish eggs, and improved the hatching rate (P<0.05). The concentration of 2.0×10 7LM-4 group treated with LM bacteria (LM-4 group) had the best treatment effect, and the average S. destruens infection rate of Xiangyun carp eggs was 26.33%, which was decreased by 45.59% compared with the CK group. The anti-S. destruens infection effect of the LM-4 test group even exceeded the positive control group (MG group) treated with malachite green, and the S. destruens infection rate of the fish eggs was decreased by 19.34% compared with the MG treatment group, and the difference was significant (P<0.05). The average hatching rate of Xiangyun carp fry in the LM-4 test group was 52.78%, which was increased by 31.46% compared with the control group, and the difference was significant (P<0.05), which was slightly lower than that of the LM-3 test group and the MG positive control group, but there was no significant difference (P>0.05).

[0080] Table 3 Effect of LM bacteria treatment on prevention and control of S. destruens in sticky fish eggs

[0081]

[0082] Note: The data in the table is represented by the average value plus or minus the standard deviation; a, b, c: multiple comparison results of S. destruens infection rate or hatching rate among the test groups, the same letter indicates no significant difference (P>0.05), and different letters indicate significant difference (P<0.05); -: does not contain this item.

[0083] It should be noted that when the present application claims involving numerical ranges, it should be understood that each numerical range of two endpoints and any number between the two endpoints can be selected. In order to prevent repetition, the present application describes the preferred embodiments.

[0084] Although the preferred embodiments of the present application have been described, those skilled in the art can make further changes and modifications to these embodiments once they know the basic inventive concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications falling within the scope of the present application.

Claims

1. A strain used to reduce saprolegniasis in fish eggs ( Saprolegnia Korean Pseudomonas (Korean Pseudomonas) infection rate and improve fish egg hatching rate Pseudomonas koreensis LM, characterized in that, It was deposited at the China Center for Type Culture Collection on November 15, 2019, with accession number CCTCC NO: M 2019934.

2. A microbial agent, characterized in that, The active ingredient of the bacterial agent includes the Korean Pseudomonas LM as described in claim 1 or the fermentation broth obtained by fermenting Korean Pseudomonas LM.

3. The application of the Korean Pseudomonas LM as described in claim 1 or the bacterial agent as described in claim 2 in aquaculture, characterized in that, The *Pseudomonas kwangsiensis* LM or its inoculum is prepared into a drug for treating pathogens in aquatic animals, wherein the aquatic animals are freshwater fish, the pathogen is *Saprolegnia*, and the concentration of *Pseudomonas kwangsiensis* LM in the drug is 2.0 × 10⁻⁶. 6 ~2.0×10 7 cfu / mL.

4. A drug, characterized in that, The drug comprises the Korean Pseudomonas LM as described in claim 1 or the bacterial agent as described in claim 2.

5. The drug according to claim 4, characterized in that, The drug also includes pharmaceutically acceptable excipients.

Images