Pseudomonas koreensis LM for inhibiting fish pathogenic fungus water mildew and application of pseudomonas koreensis LM

Abstract

The invention relates to the technical field of microorganisms, in particular to a pseudomonas koreensis LM for inhibiting fish pathogenic fungus water mildew and application of the pseudomonas koreensis LM. The pseudomonas koreensis LM is classified and named as Pseudomonas koreensis and is preserved in the China Center for Type Culture Collection on November 15, 2019, and the preservation number of the pseudomonas koreensis LM is CCTCC (China Center for Type Culture Collection) NO: M 2019934. The Korean pseudomonas LM provided by the invention has good inhibitory activity on hypha growth and spore germination of fish pathogenic fungi saprolegnia, is stable in bacteriostatic activity and good in biological safety, and has a potential value of being developed into a green and environment-friendly saprolegnia biological prevention and control agent.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a strain of Korean Pseudomonas LM capable of inhibiting fish pathogenic fungus Saprolegnia and application thereof. Background Art

[0002] Saprolegnia Saprolegnia ) is a conditionally pathogenic fungus widely found in water bodies. It can infect freshwater fish eggs, fry and adult fish, and spread the infection in the form of zoospores. Because it has no strict selectivity for hosts, can produce biofilm resistance and secrete effector proteins to interfere with host epidemic prevention, the prevention and control of Saprolegniasis has become one of the most difficult problems in fish diseases. Malachite green, a specific drug for the prevention and control of Saprolegniasis, was listed as a banned fishery drug worldwide in 1992 due to its "three-hazard" (carcinogenic, teratogenic, and mutagenic) effects. However, due to the lack of effective drugs for the prevention and control of Saprolegniasis, malachite green has been banned repeatedly, posing a great threat to the quality and safety of aquatic products. It is imperative to develop new, efficient and safe alternatives to malachite green to ensure the quality of aquatic products and protect the ecological environment of water bodies.

[0003] The aquatic animal disease prevention and control method based on biological antagonism has become a research hotspot in the aquatic field because of its green and environmental protection. The bacterial genera that have been reported to have antagonistic effects on Saprolegnia include: Aeromonas intermedius ( Aeromonas media ), Serratia marcescens ( Serratia marcescens ), Bacillus ( Bacillus ), Streptomyces ( Streptomyces )wait.

[0004] The optimum temperature range for the growth and reproduction of Saprolegnia is 5-26 ℃. Existing Saprolegnia antagonistic bacteria are mostly 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 optimum temperature range for the growth and reproduction of Saprolegnia. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a strain of Korean Pseudomonas LM for inhibiting fish pathogenic fungus Saprolegnia and application thereof.

[0006] One of the purposes of the present invention is to provide a strain of Pseudomonas koreaensis ( Pseudomonas koreensis )LM, deposited in China Center for Type Culture Collection on November 15, 2019, with the deposit number CCTCC NO: M 2019934.

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

[0008] The currently reported Saprolegnia antagonists, such as Bacillus and Streptomyces, are mostly isolated and screened from soil and aquaculture water environments. The present invention takes a different approach and successfully isolates and screens a new Saprolegnia antagonist from Saprolegnia epiphytes. The growth and reproduction temperature range of Saprolegnia is 5-26 ℃. What is valuable is that the Korean Pseudomonas LM of the present invention can grow under 4 ℃ conditions and still has good inhibitory activity against Saprolegnia at a low temperature of 15 ℃, which provides a scientific basis for the direct application of LM to aquaculture water to prevent and control the infection and spread of Saprolegnia.

[0009] The results of the test on the resistance of LM bacteria to Saprolegniasis infection of sticky fish eggs showed that LM bacteria had better resistance to Saprolegniasis infection than the positive control (MG) with the addition of malachite green. It is expected to replace malachite green for the prevention and control of Saprolegniasis in the artificial hatching of sticky fish eggs and reduce drug residues in water.

[0010] The second object of the present invention is to provide a bacterial agent, the active ingredient of the bacterial agent includes the Korean Pseudomonas LM or the fermentation liquid obtained by fermenting Korean Pseudomonas LM.

[0011] The third object of the present invention is to provide the application of the Korean Pseudomonas LM or the bacterial agent in aquatic animal breeding.

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

[0013] Furthermore, the aquatic animals include fish.

[0014] Furthermore, the fish is a freshwater fish.

[0015] Furthermore, the pathogen is a fungus.

[0016] Furthermore, the fungus includes Saprolegnia.

[0017] A fourth object of the present invention is to provide a medicine, which comprises the Korean Pseudomonas LM or the bacterial agent.

[0018] Furthermore, the drug also includes pharmaceutically acceptable excipients, such as diluents (water), lyoprotectants (mannitol, trehalose, lactose, sucrose, glycerol, amino acids), and pH regulators (sodium bicarbonate, citric acid).

[0019] The present invention has the following beneficial effects: The Korean Pseudomonas LM of the present invention has stable genetics of Saprolegnia activity and is stable to acid and alkali. The inhibition rate of Saprolegnia mycelium growth is about 70%, and the diameter of the inhibition zone of Saprolegnia spore germination is more than 30 mm. The Korean Pseudomonas LM has no infection effect on the skin of mammals and is non-pathogenic to fish. The concentration is 2.0×10 7 The infection rate of sticky fish eggs in the experimental group treated with LM bacteria with cfu / mL decreased by 45.59% compared with the control group (CK) and 19.34% compared with the positive control group (MG), and the difference was significant (P<0.05). The emergence rate of fry increased by 31.46% compared with the control group (CK) (P<0.05), and there was no significant difference with the positive control group (P>0.05). Korean Pseudomonas LM has good inhibitory activity on the growth of mycelium and spore germination of fish pathogenic fungi Saprolegnia, and the antibacterial activity is stable and the biosafety is good. It has the potential value of being developed as a green and environmentally friendly Saprolegnia biological control agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 These are the results of separation and purification of Saprolegnia pathogenic bacteria and their epiphytes, among which, A: Saprolegnia and epiphytes from Carassius auratus were transferred to PDA plates; B: Saprolegnia and epiphytes from Mandarin fish were transferred to PDA plates; C: purified Saprolegnia epiphyte JM from Carassius auratus; D: purified Saprolegnia epiphyte LM from Mandarin fish; E: purified Saprolegnia pathogen JM19 from Carassius auratus.

[0021] Figure 2 The inhibitory effects of Saprolegnia epiphytes LM and JM from different sources on Saprolegnia pathogen JM19 at different temperatures, where A: the results of confrontational growth of epiphytes and Saprolegnia at 15°C; B: the results of confrontational growth of epiphytes and Saprolegnia at 25°C.

[0022] Figure 3 Figure 5 is the genetic stability test result of LM bacteria's inhibitory activity against Saprolegnia, where A: inhibitory effect of LM bacteria of different generations (CK is the blank control of Saprolegnia pathogen JM19; G1, G5, G10 represent the confrontation growth effect of the 1st, 5th, and 10th generations of LM bacteria against Saprolegnia pathogen, respectively; B: comparison of inhibitory activity of LM bacteria of different generations (G1, G5, G10 represent the inhibitory activity of LM bacteria of the 1st, 5th, and 10th generations, respectively; a: the same letters indicate no significant difference in inhibitory activity of Saprolegnia between generations, P>0.05).

[0023] Figure 4The results of acid-base stability test of LM bacteria's inhibitory activity against Saprolegnia, where A: inhibitory effect of LM bacteria on the growth of Saprolegnia mycelium under different pH conditions; B: inhibitory effect of LM bacteria on the germination of Saprolegnia spores under different pH conditions; C: comparison of inhibition rate of LM bacteria on the growth of Saprolegnia mycelium after different pH treatments (a: the same letters indicate that there is no significant difference in the inhibitory activity of LM bacteria against Saprolegnia under different pH conditions, P>0.05); D: comparison of inhibition rate of LM bacteria on the germination of Saprolegnia spores under different pH conditions (a: the same letters indicate that there is no significant difference in the inhibitory activity of LM bacteria against Saprolegnia spores under different pH conditions, P>0.05).

[0024] Figure 5 The results of thermal stability test of LM bacteria's inhibitory activity against Saprolegnia, where A: inhibitory effect of LM bacteria on the growth of Saprolegnia hyphae after heat treatment at different temperatures (1: LM bacteria solution after heat treatment at different temperatures; 2: LM original bacteria solution control before heat treatment); B: inhibitory effect of LM bacteria on the germination of Saprolegnia spores after heat treatment at different temperatures; C: comparison of inhibitory activity of LM bacteria on the growth of Saprolegnia hyphae after heat treatment at different temperatures (a, b: t-test results of inhibition rate of each group, the same letters indicate no significant difference, P>0.05, different letters indicate significant difference, P<0.05); D: comparison of inhibitory activity of LM bacteria on the germination of Saprolegnia spores after heat treatment at different temperatures (a, b: t-test results of inhibition zone size of each group, the same letters indicate no significant difference, P>0.05, different letters indicate significant difference, P<0.05).

[0025] Figure 6 These are the results of the LM bacteria sensitivity test on mammalian skin.

[0026] Figure 7 These are the results of LM bacterial morphology observation, where A: LM bacterial colony morphology; B: LM bacterial Gram staining microscopic examination results.

[0027] Figure 8 The molecular biological identification results of LM bacteria, where A: gel electrophoresis detection results of 16S rDNA PCR amplification products of LM bacteria; B: phylogenetic tree of 16S rDNA of LM bacteria.

[0028] Fig. 9 This is a test picture of the effect of cutting mesh for artificial insemination of Xiangyun carp eggs for LM bacteria against water mold infection. DETAILED DESCRIPTION

[0029] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, 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, unless otherwise specified, can be obtained from commercial sources.

[0030] Example 1: Isolation and identification of Saprolegniasis-inhibiting bacteria.

[0031] 1. Test methods 1.1 Sample collection On March 15, 2019, one live crucian carp slightly infected with Saprolegnia and three mandarin fish that were seriously infected with Saprolegnia and had died recently were collected from the aquatic training base of Hunan University of Arts and Sciences. The Saprolegnia parasitizing the crucian carp was located on the abdomen between the left and right pectoral fins of the fish, and the hyphae were white and cotton-like, up to 2 cm long; the infected area of ​​the mandarin fish was more than 80%, and the hyphae were shorter and brownish yellow.

[0032] 1.2 Preparation of culture medium LB medium: tryptone (1%), yeast extract (0.5%), NaCl (1%), agar powder (1.8%, added when making solid culture medium); PDA plate: potato (20%), glucose (2%), agar powder (2%); test tube slant: its formula is the same as that of PDA plate.

[0033] 1.3 Isolation and purification of Saprolegnia pathogens and their epiphytic bacteria On the clean bench, the mold hyphae on the surface of the green crucian carp and mandarin fish with saprolegniasis were repeatedly rinsed with sterile water for 4-5 times, and the saprolegnia was inoculated to the center of the PDA plate with an inoculation needle. The plate was cultured at 25℃ for 48h. It was observed that the saprolegnia hyphae from the green crucian carp and mandarin fish grew radially from the center to the edge on the PDA plate, but as the culture time increased, the outer hyphae were hydrated and died. Microscopic examination revealed that there were a large number of epiphytic bacteria in the saprolegnia hyphae. The pathogenic fungus Saprolegnia was purified by the agar transfer method. Before the saprolegnia hyphae were hydrated, a fresh saprolegnia block was punched from the edge of the strain separation plate and transferred to the center of a new PDA plate. It was cultured at 15℃ and transferred repeatedly 2-3 times to obtain pure saprolegnia, which was then transferred to the PDA plate for constant temperature culture at 25℃ for 5d and stored in a refrigerator at 4℃ for later use. The epiphytic bacteria were isolated and purified by streak separation method. Bacteria were inoculated from the center of the PDA culture plate to the LB plate, and the streak purification was repeated 2-3 times. The purified strains were transferred to the slant of the PDA test tube and stored in a refrigerator at 4°C for future use.

[0034] 1.4 Screening of Saprolegniasis-Inhibiting Bacteria The isolated and purified Saprolegnia pathogen was used as the test bacteria. The Saprolegnia block was transferred to the center of the PDA plate by the agar transfer method. The purified epiphytic bacteria were inoculated 1 cm away from the Saprolegnia block. The plates were grown at 15 ℃ and 25 ℃ for 4 d, respectively. The bacteria with obvious inhibitory effect on the growth of Saprolegnia were selected and named LM, hereinafter referred to as LM bacteria.

[0035] 1.5 Stability test of LM antibacterial activity Genetic stability test: LM bacteria were continuously propagated for 10 generations using PDA slants, with the 1st, 5th and 10th generations as the tested generations, and the Oxford cup method was used to detect the genetic stability of its antibacterial activity. The Saprolegnia block was inoculated to the center of the PDA plate using the agar transfer method, and 4 Oxford cups were placed at equal intervals about 2 cm from the center. 80 μL of LM bacteria fermentation liquid of different generations (25℃, 120 r / min shaking culture for 24 h) were added respectively, and cultured at 25℃ for 48 h. The growth diameter of Saprolegnia was observed and measured, and the blank culture solution was added as the control to calculate the inhibition rate. Three parallels were set for each group. Inhibition rate = (diameter of Saprolegnia in the control group - diameter of Saprolegnia in the test group) / diameter of Saprolegnia in the control group.

[0036] Thermal stability test: The LM bacteria fermentation broth was placed in a water bath at 40 ℃, 60 ℃, 80 ℃, and 100 ℃ for 1 h, and the Oxford cup method was used to detect its thermal stability against the growth of Saprolegnia mycelium and spore germination with the inhibition rate and the size of the inhibition zone as the evaluation indicators.

[0037] Acid-base stability test: The original pH value of the LM fermentation broth was 5.0 as measured by pH test paper, and then the pH value of the fermentation broth was adjusted to 7.0 and 9.0 respectively with 1 mol / L NaOH solution. After standing at room temperature for 1 h, the alkaline-treated samples were adjusted back to the original pH value of the fermentation broth with 1 mol / L HCl solution. The Oxford cup method was used to detect the activity of the samples treated with different treatments in inhibiting the growth of Saprolegnia mycelium and spore germination.

[0038] 1.6 Biosafety Testing Skin sensitivity test: SPF mice weighing 18-22 g (purchased from Hunan Slake Jingda Experimental Animal Co., Ltd.) were used as test subjects, two males and two females, divided into two groups (divided into experimental group and control group), one male and one female in each group. The mice in the experimental group were fixed to a wooden board with a rubber band, and the hair on their abdominal skin was removed. LM bacteria were evenly applied to their exposed skin with a cotton swab from the plate. After 20 minutes of infection, the mice were released to move freely. The control group only removed the hair on the abdomen, and the mice were released after 20 minutes. The mice in the experimental group and the control group were raised separately. During the experiment, the mice were fed with special maintenance feed and free drinking water. The experimental site, cages and water utensils were cleaned and disinfected on time. Observe for 3 days, and take photos to record the skin conditions of the mice.

[0039] Fish challenge test: Crucian carp with an average body length of 8.5 cm and a body 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 was injected with 0.1 mL PBS in the thoracic cavity, and each fish in the LM injection group was injected with 0.1 mL of bacteria with a concentration of 1×10 8cfu / mL of bacterial solution, and the LM immersion group directly added bacterial solution into the basin to make the LM bacterial concentration reach 1.0×10 7 cfu / mL. Observe for 16 days without feeding and add appropriate amount of new water every 2 days. Observe and record the activity and survival of fish.

[0040] 1.7 Species identification LB plate culture was used to observe the colony morphology of LM bacteria; Gram staining was used to observe the bacterial morphology; and bacterial microbiological identification tubes (purchased from Hangzhou Microbiological Reagent Co., Ltd.) were used for physiological and biochemical identification. The molecular biological identification was carried out by 16S rDNA sequence analysis. The specific operation was as follows: LM bacterial genomic DNA was used as a template, 27 F / 1492 R was used as primers, and PCR amplification of its 16S rDNA was performed. The PCR reaction system (50 µL) was: ddH 2 O 20.0 µL, 27F 2.0 µL, 1492R 2.0 µL, DNA template 1.0 µL, 2×Taq PCR Master Mix 25.0 µL. The reaction program was: first 94 ℃ 5 min, then 94 ℃ 30 s, 58 ℃ 45 s, 72 ℃ 2 min, 30 cycles, and finally 72 ℃ 10 min. The PCR product was purified by gel recovery and sent to Shanghai Bioengineering Company for sequencing. The sequence was aligned using BLAST on the NCBI official website, and the phylogenetic tree was constructed using MEGA 5.2 software.

[0041] 2. Test results 2.1 Isolation and purification results of Saprolegnia pathogenic bacteria and their epiphytic bacteria The Saprolegnia cyanobacteria from the green crucian carp continuously grew radially from the center to the edge on the PDA plate. As the culture time increased, the outer hyphae of the Saprolegnia cyanobacteria became hydrated and dead, and became a paste ( Figure 1 A), microscopic examination revealed the presence of epiphytic bacteria in Saprolegnia mycelia. The mycelia of Siniperca chuatsi showed similar phenomena on the PDA plate, in addition, its epiphytic bacteria produced brown-yellow pigments ( Figure 1 B). Further separation and purification yielded two epiphytic bacteria strains JM ( Figure 1 C) and LM ( Figure 1 D), and a Saprolegnia pathogen JM19 ( Figure 1 E).

[0042] 2.2 Screening results of Saprolegniasis-inhibiting bacteria The Saprolegnia pathogen JM19 was used as the test object and grown against the epiphytic bacteria JM and LM at 15℃ and 25℃ for 48 hours. The results showed that LM bacteria showed good inhibitory activity against Saprolegnia, with obvious transparent circles around the bacteria, regardless of whether it was cultured at 15℃ or 25℃. The inhibitory activity was higher at 25℃, while the epiphytic bacteria JM did not have any inhibitory effect against Saprolegnia. Figure 2 ).

[0043] 2.3 Results of stability test of LM antimicrobial activity Using Saprolegnia pathogen JM19 as the target bacteria, the Oxford cup method was used to detect the genetic stability, acid-base stability and thermal stability of the antibacterial activity of LM bacteria. The results are shown in Figure 3-Figure 5 .

[0044] Depend on Figure 3 It can be seen that the fermentation broth of the 1st, 5th and 10th generations of LM bacteria has a significant inhibitory effect on the tested Saprolegnia pathogens ( Figure 3 A), the inhibition rate of LM bacteria of each generation on the growth of Saprolegnia mycelium was 60%~70%, and there was no significant difference in the inhibition rate between generations (P>0.05, Figure 3 B), indicating that the anti-saprolegnia activity of LM bacteria has good genetic stability.

[0045] Depend on Figure 4 It can be seen that the LM fermentation broth had a significant inhibitory effect on the growth of Saprolegnia mycelium under the conditions of pH 5, 7, and 9 ( Figure 4 A), the inhibition rate was more than 65%, and there was no significant difference in the inhibition rate under different pH conditions (P>0.05, Figure 4 C). LM bacteria also had a significant inhibitory effect on the germination of Saprolegnia spores under pH conditions of 5, 7, and 9 ( Figure 4 B), the diameters of the inhibition zones were all above 32 mm, and there was no significant difference in the sizes of the inhibition zones under different pH conditions (P>0.05, Figure 4 D). This indicates that the anti-saprolegnia activity of LM bacteria is stable to acid and alkali.

[0046] Depend on Figure 5 It can be seen that after heat treatment at 40℃, the LM fermentation liquid still has good antagonistic activity against the mycelial growth of Saprolegnia pathogens ( Figure 5 A), the antibacterial rate reached 71.3%, and the antibacterial activity of the fermentation broth was not significantly different from that of the control group (cultured at 25℃) (P>0.05). After the temperature was raised to above 60℃, LM bacteria lost the activity of inhibiting the growth of Saprolegnia mycelium ( Figure 5 C); After heat treatment at 40℃, the fermentation liquid of LM bacteria also had a significant inhibitory effect on the germination of Saprolegnia spores ( Figure 5B), the average diameter of the inhibition zone was 31.33 mm, which was not significantly different from the control group (P>0.05), while the antagonistic activity of LM bacteria against the germination of Saprolegnia spores after heat treatment at a temperature above 60°C was completely lost, which was significantly different from the control group (P<0.05, Figure 5 D). This indicates that the anti-saprolegnia activity of LM bacteria can remain stable below 40℃.

[0047] 2.4 Results of LM bacteria biosafety test In the skin sensitivity test, mice in both the test group and the control group did not show symptoms of infection such as skin redness, swelling and ulceration. Compared with the control group, mice in the test group had no obvious special symptoms, indicating that LM bacteria have no infection effect on the skin of mammals ( Figure 6 ).

[0048] The fish challenge test showed that there was no difference in the activity and survival of black crucian carp compared with the control group, regardless of whether the LM bacteria were immersed or injected (Table 1), indicating that LM bacteria are not pathogenic to fish.

[0049] Table 1 Results of LM bacteria challenge test on Crucian carp Note: PBS: 0.7% phosphate-balanced saline.

[0050] 2.5 LM bacterial species identification results LM colonies are round, brownish yellow, with neat edges and a smooth and moist surface; Gram-negative bacilli ( Figure 7 ); The results of physiological and biochemical identification showed that LM bacteria can utilize glucose, xylose, urea, β-galactoside, esculin, ornithine decarboxylase, lysine decarboxylase, arginine decarboxylase, have catalase and oxidase, grow at low temperature of 4℃, and cannot grow at 37℃ (Table 2), which is basically consistent with the characteristic description of Pseudomonas in Bergey's Manual of Systematic Bacteriology.

[0051] Table 2 Physiological and biochemical identification results of LM bacteria Note: +: positive test result, -: negative test result, ±: weakly positive test result.

[0052] Analysis of the 16S rDNA gene sequence (shown in SEQ ID NO.1) showed that LM bacteria Pseudomonas koreensis strain Ps 9-14 have a similarity of up to 98.88% and are clustered in one cluster on the phylogenetic tree ( Figure 8 ), the closest relative, was identified as Korean Pseudomonas ( Pseudomonas koreensis), named LM, and deposited in China Center for Type Culture Collection on November 15, 2019, with the deposit address: Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M 2019934.

[0053]

[0054] Example 2: Test on the resistance of sticky fish eggs to Saprolegnia infection by treating them with LM bacteria at different concentrations.

[0055] 1. Test methods The artificial fertilization fish egg mesh with evenly distributed Xiangyun carp was used as the experimental material (gift from Yang Fuzhong, chief technical engineer of Changde Base of Xiangyun Biotechnology Co., Ltd.). It was cut into small square mesh pieces with a side length of about 6 cm. The total number of eggs and the number of fertilized eggs were counted and inserted into the hatching cup (upper diameter 7.5 cm, bottom diameter 5.5 cm, height 7 cm) ( Fig. 9 ). Add an appropriate amount of LM bacterial fermentation broth (25 ℃, 120 r / min shaking culture for 40 h) to the incubation cup and set 2.0×10 4 cfu / mL, 2.0×10 5 cfu / mL, 2.0×10 6 cfu / mL, 2.0×10 7 cfu / mL, with a total of 4 LM bacteria concentration treatment test groups, the incubation cup without LM bacteria was used as the control group (CK group), and the incubation cup with LM bacteria added was used as the control group (CK group). 3 The malachite green hatching cup was used as the positive control group (MG group), and each group was replicated three times. The hatching cup was placed in a constant temperature incubator at 25 °C with a tray, and the number of eggs infected with Saprolegnia was counted after incubation for 48 hours. The incubation continued for 1-2 days until all the fry were hatched, and the number of fry in each cup was counted. The infection rate of Saprolegnia and the fry emergence rate of each group were calculated according to the following formula.

[0056] (1) Fish egg Saprolegnia infection rate = number of fish eggs infected by Saprolegnia on a small mesh / total number of fish eggs on the mesh.

[0057] (2) Fry emergence rate = number of fry hatched in the hatching cup / number of fertilized eggs on the small mesh in the cup.

[0058] 2. Test results The effects of different concentrations of LM bacteria on the resistance of sticky fish eggs to Saprolegnia infection are shown in Table 3. Compared with the control group (CK) without LM bacteria, the four concentrations of LM bacteria treatment test groups significantly reduced the infection rate of Saprolegnia infection in Xiangyun carp eggs and increased their hatching germination rate (P < 0.05). 7The treatment effect of LM bacteria with cfu / mL (LM-4 group) was the best. The average infection rate of Saprolegnia in Xiangyun carp eggs was 26.33%, which was 45.59% lower than that of CK group. The anti-Saprolegnia infection effect of LM-4 test group even exceeded that of the positive control group (MG group) with malachite green added. The infection rate of Saprolegnia in fish eggs was 19.34% lower than that of MG treatment group, and the difference was significant (P < 0.05). The average emergence rate of Xiangyun carp fry in LM-4 test group was 52.78%, which was 31.46% higher than that of the control group, and the difference was significant (P < 0.05). It was slightly lower than that of LM-3 test group and MG positive control group, but there was no significant difference (P > 0.05).

[0059] Table 3 Effect of different concentrations of LM bacteria on the control of Saprolegnia in sticky fish eggs Note: The data in the table are expressed as mean value plus or minus standard deviation; a, b, c: multiple comparison results of Saprolegnia infection rate or emergence rate among the experimental groups, the same letters indicate no significant difference (P>0.05), different letters indicate significant difference (P<0.05); -: does not include this item.

[0060] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0061] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

Claims

1. A Korean Pseudomonas strain that inhibits the fish pathogenic fungus Saprolegnia Pseudomonas koreensis )LM, characterized in that It was deposited in the China Center for Type Culture Collection on November 15, 2019, with the deposit number CCTCC NO: M2019934.

2. A bacterial agent, characterized in that: The active ingredient of the bacterial agent includes the Korean Pseudomonas LM according to claim 1 or a fermentation liquid obtained by fermenting the Korean Pseudomonas LM.

3. Use of the Korean Pseudomonas LM according to claim 1 or the bacterial agent according to claim 2 in aquatic animal breeding.

4. The use according to claim 3, characterized in that: The Korean Pseudomonas LM or bacterial agent is prepared into a drug for resisting pathogenic bacteria of aquatic animals.

5. The use according to claim 4, characterized in that: The aquatic animals include fish.

6. The use according to claim 5, characterized in that: The fish is a freshwater fish.

7. The use according to claim 6, characterized in that: The pathogen is a fungus.

8. The use according to claim 7, characterized in that: The fungi include Saprolegnia.

9. A drug, characterized in that The medicine comprises the Korean Pseudomonas LM according to claim 1 or the bacterial agent according to claim 2.

10. The drug according to claim 9, characterized in that The drug also includes pharmaceutically acceptable excipients.

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