A freshwater aquaculture-derived Pseudomonas bacterium capable of inhibiting and killing parasitic Saprolegnia

Abstract

The present invention relates to a freshwater aquaculture source Pseudomonas for inhibiting and killing parasitic Saprolegnia. The freshwater aquaculture source Pseudomonas is Pseudomonas soli LTBXJ, and the deposit number is CGMCC NO.24225. The strain of the present invention can destroy the mycelial tube wall and spore cyst wall of Saprolegnia, causing the mycelium to fall off and die, and the mycelium and spores in the cyst to escape and die in large quantities, thereby achieving efficient inhibition and even killing of the growth of parasitic Saprolegnia, a major pathogen of fish in my country. Since Pseudomonas is a common bacterium in freshwater aquaculture environment, the reasonable use of the bacterium will not cause harm to the aquaculture environment, and has broad application prospects.

Description

Technical Field

[0001] The invention belongs to the field of freshwater aquaculture, and particularly relates to a freshwater aquaculture-derived pseudomonad capable of inhibiting and killing parasitic Saprolegnia. Background Art

[0002] Saprolegnia parasitica is widely distributed in aquaculture waters and is an important pathogen facing the global aquaculture industry. It can infect many farmed species of freshwater fish, crustaceans, amphibians and other species. Fish with body surface damage and fish eggs in the hatching process are particularly susceptible to infection. Saprolegnia parasitica is also a major fish pathogen in my country. This study showed that a strain of Pseudomonas soli isolated from Zhejiang aquaculture freshwater has a strong inhibitory effect on Saprolegnia parasitica and has the potential to prepare a new type of green Saprolegnia preparation, which can provide an option for the withdrawal of chemical drugs.

[0003] It is generally believed that Pseudomonas inhibits the growth of pathogenic microorganisms by secreting one or more antimicrobial substances, and has the potential to become a new type of biological control drug. Studies have shown that the supernatant of sterile cell disruption of Pseudomonas has a significant inhibitory effect on the growth of Saprolegnia mycelium on confrontation plates, with a minimum effective inhibitory concentration of 6.25 mg / mL for mycelium growth and a minimum effective inhibitory concentration of 3.125 mg / mL for spore germination. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a freshwater aquaculture-derived Pseudomonas bacterium capable of inhibiting and killing parasitic Saprolegnia, which can effectively inhibit and even kill the parasitic Saprolegnia of fish pathogens in my country.

[0005] The invention provides a freshwater aquaculture-derived Pseudomonas capable of inhibiting and killing parasitic Saprolegnia. The freshwater aquaculture-derived Pseudomonas is Pseudomonas soli LTBXJ, and its preservation number is CGMCC NO.24225.

[0006] The nucleotide sequence of the Pseudomonas sp. LTBXJ is shown in SEQ ID NO.1.

[0007] The method for inhibiting the parasitic Saprolegnia by using Pseudomonas LTBXJ is as follows: the Pseudomonas LTBXJ bacterial solution after shaking culture is spread on a PDA plate to form a bacterial strip, placed in a constant temperature incubator at 28° C. for culture for 24 hours, and then placed in a constant temperature incubator at 20° C. for culture opposite to the parasitic Saprolegnia block.

[0008] The method for inhibiting and killing the parasitic Saprolegnia by Pseudomonas LTBXJ is aseptically aspirating the bacterial solution of Pseudomonas LTBXJ diluted with sterile water, adding it to a 6-well plate, then aseptically cutting a PDA agar block full of parasitic Saprolegnia hyphae and putting it into the bacterial solution, and co-culturing in a constant temperature incubator at 20°C.

[0009] The shaking culture temperature is 28-30° C., the shaking speed is 150-200 r / min, and the shaking culture time is 15-20 h.

[0010] The observation time length of the confrontation between the Pseudomonas LTBXJ and the Saprolegnia parasitica plates is based on whether the control plate is fully grown or nearly fully grown by the Saprolegnia parasitica.

[0011] Pseudomonas soli LTBXJ was deposited in the General Microbiology Center of China Microorganism Culture Collection on December 31, 2021. The deposit address is Beijing, China. Its abbreviation is CGMCC and the deposit number is CGMCCNO.24225.

[0012] Beneficial Effects

[0013] The strain of the invention is separated from a freshwater fish breeding environment, is a normal bacteria in the breeding water body, and will not cause harm to the surrounding environment and ecological balance when used reasonably, and complies with biosafety regulations; the strain can effectively inhibit or even kill the parasitic Saprolegnia of fish pathogens in my country, and the inhibition rate of plate mycelium growth can reach 76.92%; during liquid co-culture, the mycelium tube wall and the spore sac wall of the Saprolegnia can be destroyed, so that the mycelium falls off and dies, and the mycelium and the spores in the sac escape in large quantities and die. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The macroscopic growth of mycelium observed after the Pseudomonas LTBXJ inhibited the growth of Saprolegnia parasitica in Example 3: A: mycelium growth after LTBXJ inhibition, B: mycelium growth of Saprolegnia parasitica in the negative control plate.

[0015] Figure 2 The following are the microscopic growth conditions of mycelium observed after the Pseudomonas LTBXJ inhibited the growth of Saprolegnia parasitica in Example 3: A: Mycelium growth conditions after LTBXJ inhibition (115×), B: Mycelium growth conditions of Saprolegnia parasitica in the negative control plate (40×).

[0016] Figure 3 The antagonistic effects of different Pseudomonas strains on Saprolegnia parasitica;

[0017] The numbers of Pseudomonas strains A to M are: PGLU31, LYH4Y2, LYH4Y, LYG21, JY3QY6, SDZWR1 (the deposited name is LTBXJ, the experimental name is SDZWR1), JLD7CK1, JL7SY3, JLY4CY2, JLM8Y1, TBMKY1, LYMS1; M: negative control.

[0018] Figure 4Figure 1 shows the killing of Saprolegnia parasitica by Pseudomonas LTBXJ after co-culture with Saprolegnia parasitica. A, B: Saprolegnia parasitica in the untreated group; C, D: Saprolegnia parasitica after LTBXJ immersion culture. DETAILED DESCRIPTION

[0019] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0020] Example 1

[0021] Isolation, Purification and Storage of Pseudomonas LTBXJ

[0022] 100 μL of aquaculture water was taken from the aquaculture pond sample, diluted 10 times with sterile water, and 100 μL was applied to the LB plate, cultured in a 28°C constant temperature incubator for 24 hours, and a single colony was picked for purification. Then, the purified single colony was picked into the liquid culture medium, and the shaker was set to 28°C and 180r / min for 16 hours. A small amount of bacterial liquid was retained for subsequent molecular identification, and the rest of the bacterial liquid was added with glycerol and stored at -80°C for a long time.

[0023] Example 2

[0024] Molecular identification of isolates

[0025] (1) Bacterial DNA extraction

[0026] Bacterial DNA was extracted according to the instructions of the bacterial genomic DNA extraction kit.

[0027] (2) PCR amplification and sequencing analysis

[0028] The bacterial rpoD gene was selected to identify and isolate bacteria, with upstream primers: PsEG30F: 5′-ATYGAAATCGCCAARCG-3′, and downstream primers: PsEG790R: 5′-CGGTTGATKTCCTTGA-3′. The PCR reaction system was set to 50 μL: PremixTaqTM 25 μL, DNA template 4 μL, upstream and downstream primers 2 μL, and ddH2O 17 μL. The reaction conditions were set to: 94°C, 5 min; 94°C, 1 min; 55°C, 1 min; 72°C, 1 min, 30 cycles in total; 72°C, 10 min. The PCR products were analyzed by agarose gel electrophoresis, with the electrophoresis setting of 120V for 30 minutes. After that, the amplified PCR products with the expected size were recovered and sent to Sangon Biotech (Shanghai) Engineering Co., Ltd. for sequencing.

[0029] Example 3

[0030] After the isolated, purified and identified Pseudomonas was cultured in liquid LB on a shaker at 28°C, 180r / min for 20h, 100μL of bacterial solution was evenly applied to a 5mm wide bacterial band on a 6cm PDA plate 1.5cm from the edge. After the bacterial solution was completely absorbed by the culture medium, it was placed in a constant temperature incubator at 28°C for 24h. An equal amount of Sabouraud culture medium was set as a negative control. A 5mm×5mm PDA agar block full of parasitic Saprolegnia mycelia was cut with a sterilized scalpel on a circle equidistant from the edge of the culture dish full of mycelia, inoculated on the other side of the PDA plate coated with the bacterial band 1.5cm from the edge, and the plate was sealed with a sealing film and placed in a constant temperature incubator at 20°C. When the mycelia of the control group is full or nearly full of the entire plate, the shortest distance from the Saprolegnia mycelia to the bacterial band was recorded, the mycelia growth inhibition rate was calculated, and the damage to the parasitic Saprolegnia mycelia was observed under an optical microscope.

[0031] Based on the plate confrontation, it can be seen that all strains have a certain degree of inhibitory effect on the growth of Saprolegnia parasitica (Figure 3). Among them, after the inhibition of Pseudomonas LTBXJ, the growth of Saprolegnia parasitica hyphae was significantly inhibited ( Figure 1 , Figure 3 F), the growth inhibition rate was as high as 76.92% (Table 1). Under the optical dissecting microscope, it can be seen that the parasitic Saprolegnia hyphae after inhibition by Pseudomonas LTBXJ have obvious damage ( Figure 2 ): Compared with the hyphae of the control group, the hyphae are shorter and adjacent hyphae are fused.

[0032] Table 1 Inhibition rate of different Pseudomonas strains on Saprolegnia parasitica

[0033] Strain number Bacterial concentration (cells / μL) Mycelium length (cm) Inhibition rate (%) PGLU31 <![CDATA[2.00×10 5 ]]> 1.7 56.41 LYH4Y2 <![CDATA[4.75×10 5 ]]> 1.3 63.89 Y4H <![CDATA[5.40×10 5 ]]> 1.1 69.44 LYG21 <![CDATA[6.20×10 5 ]]> 1.8 53.85 J3QY6 <![CDATA[2.02×10 5 ]]> 2.9 36.96 SDZWR1 <![CDATA[2.35×10 5 ]]> 0.9 76.92 JLD7CK1 <![CDATA[1.49×10 5 ]]> 1.6 58.97 JL7SY3 <![CDATA[1.01×10 5 ]]> 1.7 56.41 JLY4CY2 <![CDATA[2.96×10 5 ]]> 2.0 48.72 JLM8Y1 <![CDATA[3.78×10 5 ]]> 2.2 43.59 TBMKY1 <![CDATA[5.20×10 5 ]]> 2.2 43.59 LYMS1 <![CDATA[3.05×10 5 ]]> 2.3 50.00 Blank control 3.9

[0034] Example 4

[0035] Aseptically pipette the Pseudomonas LTBXJ bacterial solution (10 2 / mL) was added into a 6-well plate, and a 5 mm × 5 mm PDA agar block covered with Saprolegnia parasitica hyphae was aseptically cut and placed into the bacterial solution, and co-cultured in a constant temperature incubator at 20°C, and the growth status of the hyphae was tracked and observed.

[0036] Depend on Figure 4 It can be seen that when the Pseudomonas LTBXJ of the present invention is co-cultured with the parasitic Saprolegnia, the mycelial tube wall and the spore cyst wall of the Saprolegnia can be destroyed, causing the mycelium to fall off and die, and the spores in the mycelium and cyst to escape and die in large quantities, while the untreated parasitic Saprolegnia mycelium grows vigorously during the same period, and the spore cyst wall and the mycelial tube wall are intact.

[0037] The above research results indicate that Pseudomonas LTBXJ or its components have the potential to be developed into biocontrol agents for Saprolegnias.

Claims

1. A freshwater aquaculture-derived Pseudomonas bacterium that inhibits and kills parasitic Saprolegnia, characterized in that: The freshwater aquaculture-derived Pseudomonas is soil Pseudomonas (Pseudomonas soli) LTBXJ, and its preservation number is CGMCC NO.24225.

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