CN110079470A - One plant of pseudomonad with bacteriostatic activity - Google Patents

Abstract

The invention discloses a strain of Pseudomonas with antibacterial activity, the bacterium belongs to the genus Pseudomonas moselii of the family Pseudomonadaceae, and the preservation number is CCTCC NO: M2019081. The Pseudomonas morganii LWB10 of the present invention has strong bacteriostatic activity, and its metabolites also have bacteriostatic activity, and can be used in the prevention and treatment of Agrobacterium tumefaciens and the preparation of Agrobacterium tumefaciens antibacterial drugs, and can be fermented to obtain its Metabolites provide a strong basis for the large-scale promotion and application of biocontrol bacteria.

Description

A strain of Pseudomonas with antibacterial activity

technical field

The invention relates to a pseudomonas, in particular to a pseudomonas with antibacterial activity.

Background technique

Agrobacterium tumefaciens (Agrobacterium tumefaciens) is an important plant pathogenic bacteria, its host range is very wide, can infect more than 1193 kinds of plants in 138 families such as peach, cherry, plum, apricot, pear and apple. After the fungus invades from the wound of the plant, it forms nodules on the rhizome, lateral roots or branches, hindering the transportation of water and nutrients by the root system, affecting the development of the root, resulting in nutrient deficiency, tree weakness, and even dead trees. In the cherry blossom breeding base in Ningbo area, root cancer is an important disease on cherry blossoms, which causes great economic losses to the breeding of cherry blossoms.

For many years, because the inspection and quarantine of cherry tree seedling root cancer has not kept up, root cancer has occurred in a large area in various places. With the growth of cultivation time, the incidence of root cancer in the same nursery increased year by year; due to the frequent circulation of cherry blossom seedlings in various places, root cancer also spread with the circulation of seedlings and became more serious. Preliminary investigations found that the average incidence rate of cherry blossoms in the main cultivation areas of Ningbo City was 30%, and the incidence rate of plants in re-emergence areas was even as high as 92% (Wang Zhilong et al., 2014). In production, before the onset of root cancer, methods such as dipping the roots with lime sulfur or irrigating the roots with streptomycin sulfate have a certain preventive effect on root cancer. However, after the onset of the disease, the control effect of this method is poor. Soil fumigation can reduce the number of Agrobacterium tumefaciens in the diseased land and reduce the probability of pathogenic infection. However, because soil fumigation not only kills the pathogenic bacteria, but also kills some other antagonistic microorganisms in the soil, which may lead to the recurrence of root cancer (Yakabe et al., 2010).

In the prevention and control of fruit tree diseases, there have been research reports on the use of antagonistic microorganisms to control fruit tree diseases. Liu Ting et al. isolated a Streptomyces lydidis strain A102, which can synthesize an active product. When the concentration of the active product is greater than 30 mg/L, it is effective against botrytis cinerea, Penicillium jujube and peach brown rot. The bacteria have strong inhibitory activity, and the inhibition rate can reach 100%. A Bacillus AiL3 isolated from mangroves can synthesize antibacterial proteins and inhibit the growth of mango anthracnose by affecting the metabolism of the bacteria. For the biological control of fruit tree root cancer, Australian scientists isolated a strain of Agrobacterium rhizogenes K84, which and its modified strains K1026 and WJK84-1 can be successfully used for the prevention and control of root cancer of stone fruit trees such as peach trees (Kerr, 1980; Copping, 2001; Wang Guanlin et al., 2004). Gao Zhilei et al. isolated two strains of Alcaligenes faecalis 51-A and 51-B from the rhizosphere of peach trees, which had a significant inhibitory effect on Agrobacterium tumefaciens ATCC 23308T. The inoculation experiments showed that the control effect on root cancer can reach more than 86% by infiltrating the antagonistic bacteria suspension on the indicator plant tomato in advance. However, the above-mentioned antagonistic bacteria and their metabolites with antibacterial effects have not yet been used in large-scale production. Therefore, screening and obtaining new biocontrol bacteria resources has important practical significance for the prevention and control of local root cancer in Ningbo.

SUMMARY OF THE INVENTION

In order to solve the problems of the technologies described above, the present invention provides a strain of Pseudomonas, which has strong antibacterial activity, can obviously inhibit Agrobacterium tumefaciens, and provides a basis for biological control and development of new antibacterial drugs.

The technical scheme of the present invention is to provide a kind of Pseudomonas with bacteriostatic activity, this bacterium belongs to Pseudomonas moselii (Pseudomonas moselii) of Pseudomonas family (Pseudomonadaceae), named: Pseudomonasmoselii LWB10. It is preserved in the China Center for Type Culture Collection, the preservation address is Wuhan University, Wuhan, the preservation date is January 24, 2019, and the preservation number is CCTCC NO: M2019081, hereinafter referred to as Pseudomonas morganii LWB10.

The Pseudomonas morgii LWB10 of the present invention is a Gram-negative bacterium, an aerobic bacterium, with a cell size of (0.5-0.8) μm×(1.5-1.8) μm, and terminal tufted flagella, as shown in FIG. 1 .

The 16S rDNA sequence length of the Pseudomonas LWB10 of the present invention is 1407bp, and the sequence is shown in SEQ ID NO:1.

Advantages and beneficial effects of the present invention: Pseudomonas morgii LWB10 of the present invention has strong antibacterial activity, and its metabolites also have antibacterial activity, which can be obtained in the prevention and treatment of Agrobacterium tumefaciens and the preparation of Agrobacterium tumefaciens antibacterial drugs At the same time, it can be fermented and produced to obtain its metabolites, which provides a strong basis for the large-scale promotion and application of biocontrol bacteria.

Description of drawings

Fig. 1 is an electron microscope picture of Pseudomonas LWB10 of the present invention.

Fig. 2 is a picture of the inhibition zone of Pseudomonas LWB10 of the present invention against Agrobacterium tumefaciens C58.

Fig. 3 is a phylogenetic tree of Pseudomonas LWB10 of the present invention.

Detailed ways

The present invention will be further described below in combination with specific embodiments.

The present invention provides a strain of Pseudomonas LWB10, belonging to Pseudomonas family (Pseudomonadaceae) Pseudomonas moselii (Pseudomonas moselii), preserved in China Center for Type Culture Collection (CCTCC), and the preservation address is Wuhan, Wuhan University, the deposit date is January 24, 2019, and the deposit number is CCTCC NO: M2019081, which is named Pseudomonas moselii LWB10 in the present invention.

Example 1

The Pseudomonas morgii LWB10 of the present invention is a Gram-negative, aerobic bacterium. The contact enzyme test was positive, the oxidase test was negative, and the result of the exercise test was motility. The bacterium was cultured in YEB medium (each liter medium contains 5g peptone, 5g sucrose, 1g yeast extract, 5g beef extract, 10g agar powder, pH adjusted to 7.0-7.2, 121 ℃ autoclaving for 20min. After sterilization, Add 2ml of the same sterilized 1M _MgSO4 to the culture medium; you can also choose to add beef extract and peptone (LB, KB medium) to grow well, to grow on MacConkey medium, and to grow normally on blood plates , forming gray-white flat moist colonies, smooth colonies, neat edges, and no hemolysis. The optimum growth temperature is 28°C, and it cannot grow at 4°C and 42°C. Culturing at 22-28°C can produce a large amount of yellow pigment, positive for glucose oxidative fermentation test (OF test), negative for hydrolyzed DNA, positive for arginine dihydrolase, negative for nitrate reduction, negative for acetamide hydrolysis, xylose, mannitol, Positive for maltose acid production.

Example 2

The separation process of Pseudomonas morganii LWB10 of the present invention is as follows: select cherry blossom plants infected with Agrobacterium tumefaciens (the collection place of this embodiment is in the campus of Ningbo City Vocational and Technical College, Yinzhou District, Ningbo City, Zhejiang Province, along the rhizosphere direction) Dig down, take about 5g of soil 10-20cm away from the surface soil, soak it in a sterile 50ml centrifuge tube with 10ml of sterile distilled water, put it on a vortex mixer and mix evenly, and fully release the microorganisms in the soil. Then Stand still for 10 minutes, draw a small amount of upper liquid in the ultra-clean bench for gradient dilution, set up five different gradients of 10 ¹ -10 ⁵ , draw 50ul of liquid for each gradient and spread it evenly on a 9cm YEB solid plate, and culture overnight at 28°C. The next day , Pick a single colony on a petri dish without fungal contamination, transfer it to a liquid YEB medium for cultivation, and obtain a single colony of different soil bacteria, which is used for the following identification process of antagonistic effects.

Identification of the antagonistic effect of soil bacteria on Agrobacterium tumefaciens: A large number of antagonistic bacteria of Agrobacterium tumefaciens in the soil can be screened by observing whether there is a zone of inhibition. The operation of the zone of inhibition test is as follows. Pour 15ml of YEB solid medium into a sterile petri dish and dry it for later use. Add 1ml of Agrobacterium tumefaciens bacterial solution with OD600 value of 1.0 to 10ml of solid YEB medium at a temperature of about 60°C, and mix well. Quickly pour the culture medium mixed with the bacterial solution into the culture dish prepared above, open the cover and blow dry. Use tweezers to carefully place a sterile filter paper disc with a diameter of 0.6 cm on the surface of the medium, absorb 10 μl of different soil bacteria with an OD600 value of 1.0, and drop it on the center of the filter paper. Use sterile ddH ₂ O as a blank control. After the liquid infiltrates the medium, carefully move the medium to 28°C for overnight culture, observe the formation of the inhibition zone the next day and take pictures to record it. After 2 days of cultivation, select the bacterial solution with a diameter of inhibition zone greater than 2cm as the candidate antagonistic bacteria. After in-depth research, the inhibition zone of the screened strain LWB10 co-cultured with Agrobacterium tumefaciens C58 for 2 days is shown in Figure 2.

Example 3

The screened bacterial strain LWB10 was carried out molecular identification according to the following steps: the genomic DNA of the bacteria was extracted by the wall-breaking method, and the international bacterial identification universal primer F8 (5'-AGAGTTTGATCCTGGCTCAG-3')/R1492 (5'-ACGGCTACCTTGTTACGACTT-3' ) for PCR amplification of genomic DNA. Then, the size of the amplified product was verified by agarose gel electrophoresis and recovered by tapping the gel. The recovered product was cloned by TA, connected to the pMD-18-T vector, and then transformed into E. coli TG1 competent cells. After overnight culture, it was screened by antibiotics And PCR amplification to select positive clones, and entrust Shanghai Sangon Bioengineering Technology Service Co., Ltd. to sequence the positive clones after expansion and cultivation.

The length of the 16S rDNA sequence of the strain LWB10 is 1407bp, and the sequence is shown in SEQ ID NO: 1. The sequencing results were compared in GenBank, and then the phylogenetic tree was constructed with software as shown in Figure 3 to determine the species relationship of the strain , it can be seen from the phylogenetic tree that the LWB10 of the present invention is an independent branch in the genus Pseudomonas. The results of homology analysis showed that the strain had the highest homology with the 16S rDNA sequence of Pseudomonas mosei, with a similarity of 99%. It was determined by mass spectrometry and compared with the database that it was Pseudomonas mosei. Combining the morphological characteristics of the bacteria, growth conditions, physiological and biochemical identification results, and mass spectrometry analysis in Example 1, it was determined that the strain LWB10 belonged to the genus Pseudomonas moselii in the family Pseudomonadaceae.

Example 4

Preparation of the fermentation product of Pseudomonas morselii LWB10: the isolated Pseudomonas morsei LWB10 was inoculated in YEB liquid medium, and cultured in a shaker at 28° C. at 250 rpm for 3 days. After 3 days, the bacterial solution was centrifuged at 10,000rpm for 10min to collect the supernatant, then extracted in portions with an equal volume of ethyl acetate, and the organic phase was extracted again with 1/10 volume of sterile distilled water. Next, the collected organic phase was placed on a rotary evaporator for concentration, and after concentration in portions, the remaining 2ml solution was blown dry completely on a nitrogen blower. Finally, 200 ul of chromatographically pure grade dimethyl sulfoxide (DMSO) was added to the dried test tube, and vortexed until completely dissolved to obtain the purified fermentation product of Pseudomonas LWB10.

Verification of the antibacterial activity of the fermentation product: the method of verification of the antibacterial activity of the fermentation product is the same as the process of identifying the antagonistic effect in Example 2, the difference is that the bacterial solution on the filter paper sheet is replaced with the extracted fermentation product, and after cultivating at 28°C for 24 hours, Observe the antibacterial activity of the fermented product on different strains of Agrobacterium tumefaciens, and measure the size of the inhibition zone. The results are shown in Table 1:

Table 1 Pseudomonas LWB10 of the present invention is to the antibacterial effect statistics of Agrobacterium tumefaciens

Test strain C58 GV3101 LAB4404 EHA105 C58C1 NT1RE LWB10 +++ +++ +++ +++ +++ +++

In Table 1, + indicates that the diameter of the inhibition zone is <10mm, ++ indicates that the diameter of the inhibition zone is 10mm-20mm, and +++ indicates that the diameter of the inhibition zone is >30mm. The Agrobacterium tumefaciens strains listed in the table have all been recorded and disclosed in the literature.

The above-mentioned antibacterial effect shows that Pseudomonas morganii LWB10 of the present invention has strong antibacterial activity to Agrobacterium tumefaciens, and a large amount of antibacterial active substances are also contained in its metabolites, which has further development and application value as a biocontrol bacterium of Agrobacterium tumefaciens .

The materials, reagents and experimental equipment involved in the embodiments of the present invention, if not otherwise specified, are all commercially available products in the field of plant protection.

The above are only preferred embodiments of the present invention, and it should be pointed out that for those of ordinary skill in the art, improvements and modifications can also be made without departing from the core technology of the present invention. Retouching should also belong to the patent protection scope of the present invention. Any changes within the meaning and scope equivalent to the claims of the present invention should be considered to be included in the scope of the claims.

sequence listing

<110> Ningbo City Vocational and Technical College

<120> A strain of Pseudomonas with antibacterial activity

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gggtgcaagc gttaatcgga attackgggc gtaaagcgcg cgtaggtggt tcgttaagtt 540

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ggtagagggt ggtggaattt cctgtgtagc ggtgaaatgc gtagatatag gaaggaacac 660

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aaggttaaaa ctcaaatgaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa 900

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Claims (10)

1. Pseudomonas having antibacterial activity, characterized in that the preservation number is CCTCC NO: M2019081, which belongs to the genus Pseudomonas moselii of the family Pseudomonadaceae. 2. Pseudomonas with bacteriostatic activity as claimed in claim 1, is characterized in that, is Gram-negative bacterium, aerobic bacterium, and cell size is (0.5-0.8) μ m * (1.5-1.8) μ m , with terminal plexus flagella, with motility. 3. the Pseudomonas with bacteriostatic activity as claimed in claim 1, is characterized in that, contact enzyme test is positive, and oxidase test is negative, and bacterium colony is off-white flat and moist, and bacterium colony is smooth, and edge is neat, without hemolysis; The optimum growth temperature is 28°C. It cannot grow at 4°C and 42°C. It can produce a large amount of yellow pigment when cultured at 22-28°C; glucose oxidation fermentation test is positive, DNA hydrolysis is negative, arginine dihydrolase is positive, and nitrate reduction Negative, negative for acetamide hydrolysis, positive for acid production from xylose, mannitol, and maltose. 4. the Pseudomonas with bacteriostatic activity as claimed in claim 1, is characterized in that, grows well on YEB medium, can grow on MacConkey medium, grows normally on blood plate. 5. The Pseudomonas with bacteriostatic activity as claimed in claim 1, characterized in that its 16S rDNA sequence is 1407bp in length, and its nucleotide sequence is as shown in SEQ ID NO:1. 6. The method for preparing the fermentation product of Pseudomonas described in any one of claims 1 to 5, characterized in that the steps comprise: (1) Pseudomonas was inoculated in YEB liquid medium and cultured on a shaker at 28°C for 3 days; (2) Then the bacterial liquid is centrifuged to collect the supernatant; (3) Extract the supernatant with an equal volume of organic solvent; (4) Concentrating and drying the extracted organic phase. 7. the method for preparing the fermentation product of Pseudomonas as claimed in claim 6, is characterized in that, the dried product that described step (4) obtains is redissolved through dimethyl sulfoxide to further purify fermentation product; Said step The rotating speed of the shaker in (1) is 200-250rpm; the centrifugation parameter in the step (2) is 8000-12000rpm for 5-15min. 8. the fermentation product preparation method of Pseudomonas as claimed in claim 6 is characterized in that, in described step (3), organic solvent is ethyl acetate and is divided into multiple extractions, and also after ethyl acetate extraction Include a step of re-extracting the organic phase with 1/10 volume of sterile distilled water. 9. The application of the Pseudomonas described in any one of claims 1 to 5 in the prevention and treatment of Agrobacterium tumefaciens and the preparation of Agrobacterium tumefaciens antibacterial drugs. 10. The application of the fermentation product obtained by the preparation method according to any one of claims 6 to 8 in the prevention and treatment of Agrobacterium tumefaciens and the preparation of Agrobacterium tumefaciens antibacterial drugs.