CN105441355A - Pseudomonas aeruginosa VIH2 strain and application thereof - Google Patents

Abstract

The invention discloses a strain of Pseudomonas aeruginosa VIH2 and its application. The strain can antagonize Ralstonia solanacearum (Ralstonia? solanacearum) through a type VI secretion system, and belongs to the field of molecular microorganisms. It is preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee, and the classification is called Pseudomonas (Pseudomonas? sp.), and the preservation number is CGMCC? No. 9075. As a Gram-negative bacterium, the strain has a type VI secretion system in its body, and it can use the type VI secretion system to inhibit the growth and reproduction of R. solanaceae through contact.

Description

A strain of Pseudomonas aeruginosa VIH2 and its application

technical field

The invention belongs to the technical field of microorganisms, and relates to a microorganism, in particular to a strain of Pseudomonas aeruginosa VIH2 and its application in antagonizing R. solanaceae through a type VI secretion system.

Background technique

Bacterial wilt, caused by Ralstonia solanacearum, is a plant disease that seriously damages the quality and yield of crops and fruits and vegetables. At present, the prevention and control of Laueria solanaceae mainly includes chemical control based on pesticides and biological control based on antagonistic bacteria that produce antibacterial chemicals. However, the application of pesticides seriously damages the soil structure and endangers human health; antibacterial substances are secondary metabolites that can only be produced under specific culture conditions, and they can easily cause pathogenic bacteria to develop drug resistance and lose their effect.

Gram-negative bacteria can use their own secretion system to secrete effector proteins outside the cell, and interact with other species of microorganisms, so as to gain a competitive advantage for themselves. Now more and more people are aware of the importance of food safety and environmental protection. Effector protein is considered as a potential way to control pathogenic bacteria because its secretion does not require specific culture conditions and is not easy to make pathogenic bacteria resistant. Type VI secretion system (type VI secretion system, T6SS) is a contact-dependent secretion system acting on prokaryotes, which can inhibit the reproduction of other species of bacteria.

In this experiment, a strain of Pseudomonas aeruginosa VIH2 was screened from the tomato rhizosphere soil of Nanjing Vegetable Institute. Through experiments, it was found that VIH2 can use the VI secretion system in the body to inhibit the growth of R. solanacearum through contact.

Contents of the invention

The purpose of the present invention is to provide a strain of Pseudomonas aeruginosa VIH2, which can use the VI secretion system in the body to inhibit the growth of R. solanaceae through contact.

Another object of the present invention is to provide the application of the above-mentioned Pseudomonas aeruginosa VIH2 in the control of tomato bacterial wilt caused by R. solanaceae.

The purpose of the present invention can be achieved through the following technical solutions:

The antagonistic bacteria Pseudomonas aeruginosa VIH2 (Pseudomonas aeruginosa VIH2) of the present invention was preserved on April 18, 2014 in the General Microbiology Center of China Microbiological Culture Collection Management Committee, and the address of the preservation unit is: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing Institute of Microbiology, Chinese Academy of Sciences; the classification name is Pseudomonas sp., and the preservation number is CGMCCNo.9075.

The colony of Pseudomonas aeruginosa VIH2 is small and pale yellow, with a moist surface and opaque. The bacteria are slender and of different lengths, sometimes in the shape of clubs or lines, arranged in pairs or short chains, and do not produce spores.

The physiological and biochemical characteristics of the Pseudomonas aeruginosa VIH2 are: Gram-negative, facultative aerobic, chemo-heterotrophic, negative for starch hydrolysis, positive for gelatin hydrolysis, and positive for citrate utilization.

The main nitrogen source used when cultivating Pseudomonas aeruginosa VIH2 of the present invention includes but not limited to peptone, yeast powder, potassium nitrate, ammonium nitrate, ammonium sulfate, urea; the main carbon source used includes but not limited to sucrose, manna Alcohol, maltose, glucose; inorganic components used include but are not limited to potassium chloride, sodium chloride, monobasic sodium phosphate, dipotassium hydrogen phosphate, calcium chloride dihydrate, magnesium sulfate heptahydrate, heptahydrate, and ferrous sulfate . The fermentation of Pseudomonas aeruginosa VIH2 in the present invention can be carried out at 30° C. and pH 7.0.

The Pseudomonas aeruginosa VIH2 of the present invention can kill R. solanaceae through contact, and after the two are contact-cultured on the NA plate for 24 hours, the biomass of R. solanaceae decreases by more than 5 orders of magnitude. If the regulatory gene ppKA of the type VI secretion system of the VIH2 strain was destroyed, the biomass of R. solanaceae decreased by less than 2 orders of magnitude after 24 hours of contact culture. This experimental result proves that Pseudomonas aeruginosa VIH2 can use the VI secretion system in vivo to inhibit the growth of R. solanaceae through contact.

Pseudomonas aeruginosa described in the present invention can effectively inhibit the colonization of R. solanaceae in tomato roots, but if the ppKA gene is destroyed, the effect of Pseudomonas aeruginosa on inhibiting the colonization of R. solanaceae Not obvious.

Pseudomonas aeruginosa described in this experiment can also effectively inhibit the growth of R. ability of bacteria.

The application of the above-mentioned Pseudomonas aeruginosa VIH2 in inhibiting the growth of R. solanaceae by means of contact by utilizing the VI secretion system in the body.

The application of the above-mentioned Pseudomonas aeruginosa VIH2 in the prevention and treatment of tomato bacterial wilt caused by R. solanaceae.

Application of the above-mentioned Pseudomonas aeruginosa VIH2 in the preparation of bacterial fertilizer for preventing and treating R. solanaceae.

Beneficial effects of the present invention:

The Pseudomonas aeruginosa VIH2 of the present invention can utilize the VI secretion system in the body to inhibit the growth of R. solanaceae through contact, and reduce the incidence of tomato bacterial wilt caused by R. solanaceae.

Description of drawings:

Fig. 1 is the bacterium colony figure of bacterial strain VIH2 of the present invention

Fig. 2 is the contact and non-contact inhibition test result of bacterial strain VIH2 of the present invention to R. solanaceae

Figure 3 shows the results of the inhibition test of VIH2 strain and VIH2△ppKA strain against R. solanaceae

Figure 4 shows the antagonistic effect of VIH2/VIH2△ppKA on R. solanaceae in soil

detailed description:

The present invention will be further described below in conjunction with the accompanying drawings.

First, prepare the following two culture media.

LB medium: tryptone 10g, yeast powder 5g, sodium chloride 10g, agar powder 15g, distilled water 1000ml, pH7.0, sterilized at 121°C for 20min.

LB liquid medium: without adding agar powder, other conditions are the same as above.

NA medium: tryptone 5g, yeast powder 0.5g, beef extract 3g, glucose 10g, agar powder 15g, distilled water 1000ml, pH7.0, sterilized at 115°C for 30min.

NA liquid medium: without adding agar powder, other conditions are the same as above.

Weigh 10g of the tomato rhizosphere soil taken from Nanjing Vegetables and put it in a 250ml Erlenmeyer flask filled with 90ml of sterilized water, and vibrate for 20min at 30°C and 150r min ⁻¹ in a shaker. Let it stand for 10 minutes to obtain a soil suspension. The soil suspension contains several kinds of antagonistic bacteria, which are diluted by the dilution method and applied to LB medium. After culturing in a 30°C incubator for 24 hours, different types of typical single colonies are picked, purified on a plate, and stored in glycerol at -70°C. stand-by.

Embodiment 1 Contact inhibition test

The contact-dependent antagonistic bacteria were screened by plate confrontation.

In order to facilitate the observation of the effect, experiments were carried out using R. solanaceae with red fluorescent markers.

Firstly, R. solanacearum was inoculated into the NA liquid test tube containing 30ppm gentamycin, cultured overnight at 180r/min at 30°C. Then take 1ml of the bacterial solution and centrifuge at 12000rpm for 2min to collect the bacterial cells, and resuspend with 1ml of sterile water for 3 times.

Experiment 1:

Take 5 μL of the resuspended bacteria solution and inoculate it on an LB plate without any antibiotics to form plaques of R. solani. Next to the plaques, use a sterilized toothpick to inoculate the isolated and purified strains, and observe the confrontation results .

Experiment 2:

Take 5 μL of the resuspended bacteria solution and spread it on an LB plate without any antibiotics, place a 0.22 μm sterile filter membrane in the center of the plate, and use a sterilized toothpick to inoculate the isolated and purified strain on the other side of the filter membrane , observe the confrontation result.

Since the 0.22 μm sterile filter membrane can pass small molecular compounds but not bacteria, the contact-dependent antagonistic bacteria have antagonistic effects under the conditions of Experiment 1 but no longer have antagonistic effects under the conditions of Experiment 2. The role of yeast.

A strain of contact-dependent antagonistic bacteria was screened out by the above method, as shown in Figure 1, the colony formed by this strain is light yellow, the surface is moist, opaque, the bacteria are slender and of different lengths, and sometimes they are club-shaped or linear Shaped, arranged in pairs or short chains, without spores, named VIH2. After the strain was cultured in contact with R. solanaceae on the NA plate for 24 hours, the biomass of R. solanaceae decreased by more than 5 orders of magnitude (Figure 2), indicating that the occurrence of antagonism depends on VIH2 and R. solanaceae contact with bacteria.

The strains isolated by the above method were screened and sequenced. According to the sequencing results of 16SrDNA, they were searched and analyzed online at http://www.ncbi.nlm.nih.gov, and homologous with other 16SrDNA sequences in GenBank using Blast software. Sexual comparison, selection of similar sequence and VIH2 sequence using MEGAversion3 software to construct VIH2 16SrDNA phylogenetic tree, combined with biolog experimental results, identified as Pseudomonas aeruginosa (Ralstonia solanacearum), homology 99%. The strain was preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee on April 18, 2014, with the preservation number CGMCCNo.9075.

Example 2

Aerobic test

Pour the sterilized LB culture medium into 3 sterilized test tubes, at about 2/3, on the aseptic operating table, use an inoculation needle to pick up the bacteria cultured on the slant, puncture and inoculate into the above culture base (must pierce to the bottom of the tube). Cultivate at 30°C, and observe the results in 3 days to 7 days respectively. Those that grow on the surface of the agar column are aerobic bacteria, and those that grow along the puncture line are anaerobic bacteria or facultative aerobic bacteria. The test result is facultative aerobic.

Starch hydrolysis test

a. Culture medium and reagents, add 0.2% soluble starch to broth peptone agar, pack into Erlenmeyer flasks, and sterilize at 121°C for 20 minutes for later use. Lugol's iodine solution: 1g of iodine tablet, 2g of potassium iodide, first dissolve potassium iodide with a small amount (3-5mL) of distilled water, then add iodine tablet, after the iodine is completely dissolved, add water to dilute to 300mL.

b. Bacteria culture and result observation, take the bacteria and spot on the plate, culture at 30°C for 2 to 4 days, after the formation of colonies, add Lugol's iodine solution dropwise on the plate, so as to cover the surrounding area of the colony, the plate It is blue, and if there is a colorless transparent circle around the colony, it means that the starch has been hydrolyzed. The size of the transparent circle generally indicates the size of the ability to hydrolyze starch.

The test result was negative for starch hydrolysis.

Gelatin hydrolysis test

a. Medium and reagents, peptone 5g, gelatin 120g, distilled water 1000mL. Adjust the pH to 7.2-7.4, divide into test tubes, the height of the culture medium is about 4-5cm, and sterilize at 121°C for 20min.

b. Bacteria culture and result observation, inoculated in the center of the test tube by puncture method. Cultivate in a 30°C incubator for one month, and observe whether the gelatin liquefies.

The test result was positive for gelatin hydrolysis.

Use of citrate

a. Medium and reagents, sodium citrate 2g, NaCl 5g, MgSO ₄ ·7H ₂ O0.2g, (NH ₄ ) ₂ ·HPO ₄ 1g, 1% Australian thymol blue aqueous solution 10mL, agar 20g, distilled water 1000mL, pH6. 8-7.0, sterilized at 121°C for 20 minutes.

b. Bacteria culture and result observation, inoculate the young bacteria on the slant, culture at 30°C for 3-7 days, the medium is alkaline (blue) as a positive reaction, and the one that remains unchanged is negative.

Test results for citrate utilization were positive.

Example 3

Among the seven protein secretion systems of Gram-negative bacteria, only the type VI secretion system is directed against prokaryotes and is contact-dependent. In order to verify whether the type VI secretion system plays a major role, we constructed a mutant strain VIH2 △ppKA. The ppKA gene of this strain is destroyed, and the ppKA gene is a key gene regulating the type VI secretion system, and the destruction of the gene means that the type VI secretion system is inactivated.

First, inoculate VIH2 strain/VIH2△ppKA strain into LB liquid test tube containing 30ppm kanamycin, culture at 180r/min, 30°C for 24 hours to prepare VIH2/VIH2△ppKA seed liquid; Inoculate into NA liquid test tube containing 30ppm gentamycin, 180r/min, and culture at 30°C for 24 hours to prepare seed liquid.

Then, draw 100 μL from the VIH2/VIH2△ppKA seed solution and inoculate it into 100ml LB liquid medium containing 30ppm kanamycin, culture at 180r/min, 30℃ until the _OD600 is about 2.5; Inoculate 100 μL of the solution into 100 ml NA liquid medium containing 30 ppm gentamicin, and culture at 180 r/min at 30°C until the OD ₆₀₀ is about 2.5.

Finally, centrifuge at 14000r/min for 3min to precipitate 100ml VIH2/VIH2△ppKA bacterial liquid, wash with sterile water for 3 times, and then resuspend with LB liquid medium; The Laueria liquid was precipitated, washed 3 times with sterile water, and then resuspended with NA liquid medium.

Experiment 1:

The suspension of VIH2 bacteria and the suspension of R. solanaceae were mixed at a ratio of 1:5.

Control group (CK): inoculate 10 μL of sterile water on the LB plate first, then cover it with a layer of 0.22 μm filter membrane, and finally inoculate 50 μL of R. solanaceae suspension on the filter membrane.

Treatment group 1: inoculate 10 μL of VIH2 bacterial suspension on LB plates first, then cover a layer of 0.22 μm filter membrane on the VIH2 bacterial suspension, and finally inoculate 50 μL of R. solanacae suspension on the filter membrane.

Treatment group 2: first put a layer of 0.22 μm filter membrane on the LB plate, then inoculate 10 μL of VIH2 suspension and 50 μL of R. solanaceae suspension on the filter membrane.

After culturing for 24 hours, the biomass change of R. solanaceae was detected by plate counting method. Through the above experiments, it was tested whether contact was required for VIH2 to inhibit R. solanacearum.

The experimental results show that, compared with the control group, the order of magnitude of R. solanaceae in treatment group 2 decreased by more than 5, and the order of magnitude of R. solanaceae in treatment group 1 decreased by less than 1 order of magnitude (as shown in Figure 2 ). The results showed that the VIH2 strain was a contact-dependent antagonistic bacteria.

Experiment 2:

Control group (CK): R. solanacearum was mixed with sterile water at a ratio of 5:1.

Treatment group 1: R. solanacii and VIH2 strains were cultured in a 5:1 mixed culture.

Treatment group 2: R. solanaceae and VIH2△ppKA strain were mixed at 5:1.

After culturing for 24 hours, the biomass change of R. solanaceae was detected by plate counting method. Through the above experiments, it was detected whether the production of antagonism was related to the type VI secretion system in VIH2 bacteria.

The experimental results show that, compared with the control group, the order of magnitude of R. solanaceae in treatment group 1 decreased by more than 5, and the order of magnitude of R. solanaceae in treatment group 2 decreased by less than 2 orders of magnitude (as shown in Figure 3 ). The results indicated that the type VI secretion system in the VIH2 strain plays a major role in antagonizing R. solanacearum.

Example 4

The present invention has antagonistic effect on R. solanacearum, which will be explained through soil in situ test below.

Collect fresh soil from the 0-20cm soil layer under the natural conditions of Nanjing Vegetable Institute, pass through a 5mm sieve, fill each beaker with 500g of soil, and sterilize.

VIH2/VIH2△ppKA bacterial suspension: Inoculate VIH2/VIH2△ppKA of the present invention in LB liquid medium, culture at 180r/min, shaker at 30°C overnight, until OD ₆₀₀ is about 2.5. Then the bacterial solution was centrifuged at 12000rpm for 5min, and then resuspended with sterile water.

KT2440 bacterial suspension: inoculate KT2440 in LB liquid medium, culture at 180r/min, shaker at 30°C overnight, until OD ₆₀₀ is about 2.5. Then the bacterial solution was centrifuged at 12000rpm for 5min, and then resuspended with sterile water.

Suspension of R. solanaceae: inoculate R. solanaceae in NA liquid medium, culture at 180r/min, shaker at 30°C overnight, until OD ₆₀₀ is about 2.5. Then the bacterial solution was centrifuged at 12000rpm for 10min, and then resuspended with sterile water.

Control group: the suspension of R. solanacii and the suspension of KT2440 bacteria were mixed at a ratio of 1:1 and added to the sterilized soil for co-cultivation.

Treatment group 1: Mix the suspension of Ralstonia solanacearus and the suspension of VIH2 bacteria at a ratio of 1:1 and add them to the sterilized soil for co-cultivation.

Treatment group 2: The suspension of R. solanacii and the suspension of VIH2△ppKA bacteria were mixed at a ratio of 1:1 and added to the sterilized soil for co-cultivation.

Then, samples were taken on the 0th, 3rd, 7th, 21st, and 35th days, and the biomass changes of R. solanaceae were detected by plate counting.

The experimental results showed that with the passage of time, the biomass of R. solanaceae decreased under the three treatments, but the magnitude of the decrease was significantly different. Compared with the control group, the biomass of R. solanaceae in treatment group 1 decreased significantly by more than one order of magnitude, while the R. solanaceae in treatment group 2 had no significant difference compared with the control group (Fig. 4). The results show that the Pseudomonas aeruginosa VIH2 invented in this experiment can effectively antagonize R. solanacearum, and the main antagonism is through the type VI secretion system in the VIH2 bacterium.

Claims (4)

1. A strain of Pseudomonas aeruginosa VIH2 was deposited on April 18, 2014 in the General Microbiology Center of China Committee for Culture Collection of Microorganisms, classified as Pseudomonas sp., and the preservation number is CGMCCNo.9075. 2. The application of Pseudomonas aeruginosa VIH2 according to claim 1 in inhibiting the growth of R. solanaceae by contacting the VI secretion system in the body. 3. The application of Pseudomonas aeruginosa VIH2 described in claim 1 in the prevention and treatment of tomato bacterial wilt caused by R. solanaceae. 4. the application of the Pseudomonas aeruginosa VIH2 described in claim 1 in the bacterial fertilizer of preparation prevention and treatment of R. solanaceae.