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

Abstract

The invention relates to a freshwater aquaculture-derived pseudomonad for inhibiting and killing Saprolegnia multiflora. The freshwater aquaculture-derived pseudomonad is Pseudomonas simiae TGYZ, and its deposit number is CGMCC NO.24227. The strain of the invention can destroy the mycelial tube wall and the spore cyst wall of Saprolegnia, causing the mycelium and the spores in the cyst to escape in large quantities and die, thereby realizing efficient inhibition and even killing of the growth of Saprolegnia multiflora, a major pathogen of fish in my country. Since Pseudomonas is a common bacterium in freshwater aquaculture environments, 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 Pseudomonas bacteria capable of inhibiting and killing Saprolegnia multiflora. Background Art

[0002] Saprolegniasis is a common fish disease in freshwater aquaculture worldwide. The pathogens are mainly Saprolegnia, Achlya and Aphanomyces. Due to the wide range of Saprolegnia hosts and rapid transmission, large-scale outbreaks of Saprolegniasis can often occur in farmed freshwater fish, their seedlings and eggs in a short period of time during low temperature seasons. Relevant studies have shown that Saprolegnia ferax is the main pathogenic species in my country. Since chemical drugs such as malachite green that are effective in preventing and controlling Saprolegniasis have been listed as banned fishery drugs, the types of green control drugs currently available are very limited. Therefore, based on the concept of biosafety, screening and developing new green antifungal preparations has always been an urgent need on the production front line.

[0003] Pseudomonas is one of the hot topics in microbial research. Currently, it is generally believed that its biological control mechanism is that it can secrete one or more antimicrobial substances to inhibit the growth of pathogenic microorganisms, thereby achieving its biological control effect. Studies have shown that Pseudoalteromonas sp. can inhibit a variety of plant pathogens through the antifungal active substances it produces. The fungicide preparation made from it has a control effect of up to 66.7% on the plant fungal disease gray mold at low temperatures of 10-15°C. However, Pseudomonas is rarely used in the control of Saprolegniasis. Summary of the invention

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

[0005] The invention provides a freshwater aquaculture-derived Pseudomonas bacteria capable of inhibiting and killing Saprolegnia multiflora. The freshwater aquaculture-derived Pseudomonas bacteria is Pseudomonas simiae TGYZ, and the preservation number is CGMCC NO.24227.

[0006] The nucleotide sequence of Pseudomonas simianus TGYZ is shown in SEQ ID NO.1.

[0007] The method for inhibiting Saprolegnia polystomata by Pseudomonas simiana TGYZ is as follows: the bacterial liquid of Pseudomonas simiana TGYZ 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 Saprolegnia polystomata block.

[0008] The method for killing Saprolegnia multisperma by Pseudomonas TGYZ is aseptically aspirating a bacterial solution of Pseudomonas TGYZ diluted with sterile water, adding the solution to a 6-well plate, then aseptically cutting a PDA agar block covered with Saprolegnia multisperma hyphae and placing the block in the bacterial solution, and co-culturing the block 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 length of observation time for the confrontation between the Pseudomonas simiana TGYZ and the Saprolegnia multiflora plates is based on the control plate being fully or nearly fully grown with Saprolegnia multiflora.

[0011] Pseudomonas simiae TGYZ was deposited in the General Microbiology Center of China Culture Collection Administration on December 31, 2021. Its abbreviation is CGMCC and the deposit number is NO.24227.

[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 fish pathogen Saprolegnia multisperma in my country, and the inhibition rate of plate hyphae growth can reach 76.32%; during liquid co-culture, the hyphae tube wall and the spore sac wall of the Saprolegnia can be destroyed, so that the hyphae 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 was observed after the growth of Saprolegnia multisperma was inhibited by Pseudomonas simiana TGYZ in Example 3: A: mycelium growth after inhibition by TGYZ, B: mycelium growth of Saprolegnia multisperma in the negative control plate.

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

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

[0017] The Pseudomonas strains A to M are numbered as follows: PGLU31, LYH4Y2, LYH4Y, LYG21, JY3QY6, JLD7CK1, JL7SY3, JLY4CY2, JLM8Y1, TBMKY1, STWRY1 (the deposited name is TGYZ, and the experimental name is STWRY1), LYMS1; M: negative control.

[0018] Figure 4 Figure 1 shows the killing of Saprolegnia multiflora by co-culture of Pseudomonas simiana TGYZ and Saprolegnia multiflora. A, B: Saprolegnia multiflora in the untreated group; C, D: Saprolegnia multiflora after immersion culture of TGYZ. 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 Preservation of Pseudomonas simianus Strain TGYZ

[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 product was analyzed by agarose gel electrophoresis, with the electrophoresis setting of 120V for 30 minutes. After that, the amplified PCR product with the expected size was 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 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 was 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 of the 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 multiflora ( Figure 3 Among them, the growth of Saprolegnia multifida hyphae was significantly inhibited after the inhibition of Pseudomonas simiana strain TGYZ ( Figure 1 , Figure 3 k), the growth inhibition rate was as high as 76.32% (Table 1). Under the optical dissecting microscope, it can be seen that the mycelium of Saprolegnia multifida after inhibition by Pseudomonas simiana TGYZ was significantly damaged ( 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 multiflora

[0033] Strain number Bacterial concentration (cfu / μL) Mycelium length (cm) Inhibition rate (%) PGLU31 <![CDATA[2.00×10 5 ]]> 1.3 71.11 LYH4Y2 <![CDATA[4.75×10 5 ]]> 1.5 60.53 Y4H <![CDATA[5.40×10 5 ]]> 1.9 50.00 LYG21 <![CDATA[6.20×10 5 ]]> 1.6 64.44 J3QY6 <![CDATA[2.02×10 5 ]]> 2.7 42.55 JLD7CK1 <![CDATA[1.49×10 5 ]]> 2.0 55.56 JL7SY3 <![CDATA[1.01×10 5 ]]> 2.3 48.89 JLY4CY2 <![CDATA[2.96×10 5 ]]> 2.1 53.33 JLM8Y1 <![CDATA[3.78×10 5 ]]> 2.2 51.11 TBMKY1 <![CDATA[5.20×10 5 ]]> 2.4 46.67 STWRY1 <![CDATA[6.70×10 5 ]]> 0.9 76.32 LYMS1 <![CDATA[3.05×10 5 ]]> 3.6 23.40 Negative control 4.5

[0034] Example 4

[0035] Aseptically pipette the Pseudomonas simianus TGYZ bacterial solution (10 2 / mL) was added into a 6-well plate, and a 5 mm × 5 mm PDA agar block covered with Saprolegnia multifida 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 simiana TGYZ of the present invention is co-cultured with Saprolegnia polysperma, the mycelial tube wall and sporangium wall of the Saprolegnia polysperma can be destroyed, causing the mycelium and spores in the sac to escape in large quantities and die, while the mycelium of the untreated Saprolegnia polysperma grows vigorously during the same period, and the sporangium wall and mycelial tube wall are intact.

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

Claims

1. A freshwater aquaculture-derived Pseudomonas bacterium that inhibits and kills Saprolegnia multiflora, characterized in that: The freshwater aquaculture-source Pseudomonas is Pseudomonas simiae TGYZ, and its preservation number is CGMCC NO.24227.

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